CpG oligonucleotide analogs containing hydrophobic T analogs with enhanced immunostimulatory activity

ABSTRACT

The invention relates to oligonucleotides including at least one lipophilic substituted nucleotide analog and a pyrimidine-purine dinucleotide. The invention also relates to pharmaceutical compositions and methods of use thereof.

FIELD OF THE INVENTION

The present invention relates generally to the field of immunology. Morespecifically the invention relates to therapeutic oligonucleotides withenhanced immunostimulatory capacity.

BACKGROUND OF THE INVENTION

Bacterial DNA has immune stimulatory affects to activate B cells andnatural killer cells, but vertebrate DNA does not (Tokunaga, T., et al.,1988. Jpn. J. Cancer Res. 79:682-686; Tokunaga, T., et al., 1984, JNCI72:955-962; Messina, J. P., et al., 1991, J. Immunol. 147:1759-1764; andreviewed in Krieg, 1998, In: Applied Oligonucleotide Technology, C. A.Stein and A. M. Krieg, (Eds.), John Wiley and Sons, Inc., New York,N.Y., pp. 431-448). It is now understood that these immune stimulatoryeffects of bacterial DNA are a result of the presence of unmethylatedCpG dinucleotides in particular base contexts (CpG motifs), which arecommon in bacterial DNA, but methylated and underrepresented invertebrate DNA (Krieg et al, 1995 Nature 374:546-549; Krieg, 1999Biochim. Biophys. Acta 93321:1-10). The immune stimulatory effects ofbacterial DNA can be mimicked with synthetic oligodeoxynucleotides (ODN)containing these CpG motifs. Such CpG ODN have highly stimulatoryeffects on human and murine leukocytes, inducing B cell proliferation;cytokine and immunoglobulin accretion; natural killer (NK) cell lyticactivity and IFN-γ secretion; and activation of dendritic cells (DCs)and other antigen presenting cells to express costimulatory moleculesand secrete cytokines, especially the Th1-like cytokines that areimportant in promoting the development of Th1-like T cell responses.These immune stimulatory effects of native phosphodiester backbone CpGODN are highly CpG specific in that the effects are dramatically reducedif the CpG motif is methylated, changed to a GpC, or otherwiseeliminated or altered (Krieg et al, 1995 Nature 374:546-549; Hartmann etal, 1999 Proc. Natl. Acad. Sci USA 96:9305-10).

In early studies, it was thought that the immune stimulatory CpG motiffollowed the formula purine-purine-CpG-pyrimidine-pyrimidine (Krieg etal, 1995 Nature 374:546-549; Pisetsky, 1996 J. Immunol. 156:421-423;Hacker et al., 1998 EMBO J. 17:6230-6240; Lipford et al, 1998 Trends inMicrobiol. 6:496-500). However, it is now clear that mouse lymphocytesrespond quite well to phosphodiester CpG motifs that do not follow this“formula” (Yi et al., 1998 J. Immunol. 160:5898-5906) and the same istrue of human B cells and dendritic cells (Hartmann et al, 1999 Proc.Natl. Acad. Sci USA 96:9305-10; Liang, 1996 J. Clin. Invest.98:1119-1129).

Several different classes of CpG nucleic acids has recently beendescribed. One class is potent for activating B cells but is relativelyweak in inducing IFN-α and NK cell activation; this class has been tamedthe B class. The B class CpG nucleic acids typically are fullystabilized and include an unmethylated CpG dinucleotide within certainpreferred base contexts. See, e.g., U.S. Pat. Nos. 6,194,388; 6,207,646;6,214,806; 6,218,371; 6,239,116; and 6,339,068. Another class of CpGnucleic acids activates B cells and NK cells and induces IFN-α; thisclass has been termed the C-class. The C-class CpG nucleic acids, asfirst characterized, typically are fully stabilized, include a Bclass-type sequence and a GC-rich palindrome or near-palindrome. Thisclass has been described in U.S. provisional patent application60/313,273, filed Aug. 17, 2001 and U.S. Ser. No. 10/224,523 filed onAug. 19, 2002 and related PCT Patent Application PCT/US02/126,468published under International Publication Number WO 03/015711.

SUMMARY OF THE INVENTION

The invention relates to an oligonucleotide which comprises one or moremodifications that elicits enhanced immunostimulatory capacity. Inparticular, the invention is based on the finding that specificsub-classes of oligonucleotides having at least one lipophilicsubstituted nucleotide analog are highly effective in mediating immuneresponse. These oligonucleotides are useful therapeutically andprophylactically for inducing an immune response and for treatingdiseases and disorders such as cancer and viral infections.

In ore aspect, the invention is a composition comprising the sequence:R₁YZR₂, wherein R₁ and R₂ represent a lipophilic substituted nucleotideanalog (L), a nucleotide, and a linkage, wherein at least one of R₁ andR₂ is a lipophilic substituted nucleotide analog (L), wherein Y is apyrimidine nucleotide and wherein Z is a purine, a pyrimidine, or anabasic residue.

In some embodiments, L comprises a 5- or 6-membered ring nucleobaseanalog.

In other embodiments of the aspect of the invention, L is a group offormula I.

having the following elements: A, B, X, D, E, and F are C (carbon) or N(nitrogen) optionally bearing hydrogen or a substituent; n is 0 or 1;the dotted lines indicate optional double bonds; wherein at least onesubstituent is not chosen from the group consisting of oxo, thio,hydroxy, mercapto, imino, amino, methyl and hydrogen, and that the totalof A, B, X, D, E and F atoms is not more than 3 nitrogens (N). In somecases n is 1, and in other cases n is 0. In some embodiments, all atomsA, B, X, D, E, F are carbon (C). In some embodiments, one, two or threeof the atoms A, B, X, D, E, F are nitrogen (N). According to someembodiments, at least one of the atoms A, B, X, D, B, F is substitutedby one of the following: F, Cl, Br, I, alkyl, alkenyl, alkinyl,halogenated alkyl, halogenated alkenyl, cycloalkyl, O-alkyl, O-alkenyl,—NH-alkyl, —N(alkyl)₂; —S-alkyl, —SO-alkyl, —SO₂-alkyl, nitro, cyano,carboxylester, phenyl, thiophenyl, benzyl, oxo, thio, hydroxy, mercapto,and imino, wherein at least one substituent is not oxo, thio, hydroxy,mercapto, imino, amino or methyl. According to yet other embodiments,one of the two atoms A or E is substituted by one of the following: F,Cl, Br, I, C₂-C₆-alkyl, alkenyl, alkinyl, halogenated alkyl, halogenatedalkenyl, cycloalkyl, O-alkyl, O-alkenyl, —N-alkyl, —N(alkyl)₂; —S-alkyl,—SO-alkyl, —SO₂-alkyl, nitro, cyano, carboxylester, phenyl, thiophenyl,benzyl, or methyl, provided that if methyl then A, B, X, D, E, and F areall C.

In some embodiments formula I comprises a substituted pyrimidine,uracil, toluene, imidazole or pyrazole or triazole. According to otherembodiments, formula I is selected from the following: 5-chloro-uracil,5-bromo-uracil, 5-iodo-uracil, 5-ethyl-uracil, 5-propyl-uracil,5-propinyl-uracil, (E)-5-(2-bromovinyl)-uracil, and2.4-difluoro-toluene. According to one embodiment of the invention,formula I is fused with a 3- to-6-membered aromatic or aliphatic ringsystem. According to other embodiments, formula I is linked to a 5- to6-membered sugar moiety, including a pentose or hexose. In some cases,the pentose is a furanose and hexose is a pyranose, which can optionallybe substituted by F, amino, alkoxy, alkoxy-ethoxy, amonipropyl, alkenyl,alkinyl, or a O2,C4-alkylene bridge In other cases, the furanose isribose or deoxyribose.

According to some embodiments of the invention, R₁ and R₂ are both L. Insome embodiments, R₁ is L and R₂ is a nucleotide. Alternatively, in somecases R₁ is a L and R₂ is a linkage, such that the oligonucleotidecomprises a structure 5′ R₁CG 3′. Other embodiments includeoligonucleotide wherein R₁ is L and R₂ is a linkage, and wherein a R₃ is5′ to R₁YZ, such that the oligonucleotide comprises a structure 5′R₃R₁YZ 3′. In some embodiments, R₁ is L and R₂ is a linkage, and whereina second R₁ is 5′ to R₁YZ spaced by one nucleotide N, such that theoligonucleotide comprises a structure 5′ R₁NR₁YZ 3′. In some cases, theoligonucleotide may include two 5′ R₁NR₁YZ 3′ motifs.

According to some embodiments, The oligonucleotide includes Y that isone of the following pyrimidines: cytosine, 5-methyl-cytosine,5-hydroxy-cytosine, 5-hydroxymethyl-cytosine, 5-halogeno-cytosine,2-thio-cytosine, 4-thio-cytosine, N3-methyl-cytosine, N4-alkyl-cytosineor a 6-substituted cytosine.

According to some embodiments, the oligonucleotide includes Z that is apurine nucleotide including; guanine, 7-deaza-guanine, hypoxanthine,7-deaza-hypoxanthine, 2-amino-purine, 4-thio-purine, 2.6-diamino-purine,8-oxo-7.8-dihydroguanine, 7-thia-8-oxo-7.8-dihydroguanine,7-allyl-8-oxo-7.8-dihydroguanine, 7-deaza-8-aza-guanine, 8-aza-guanine,N1-methyl-guanine or purine. In other embodiments, Z is a pyrimidinenucleotide, including T.

According to some embodiments of the invention, R₂ is L and R₁ is anucleotide.

According to some embodiments, the oligonucleotide is between 3-100nucleotides in length; for example, the oligonucleotide is 3-6nucleotides in length, 3-100 nucleotides in length, or 7-100 nucleotidesin length. In some circumstances, the oligonucleotide is T-rich, suchthat at least 80% of the nucleotides are T.

The invention includes embodiments comprising at least one palindromicsequence. For example, in some cases, the oligonucleotide includes twopalindromic sequences.

According to the invention, some embodiments include one to fourunmethylated CG dinucleotides. In some embodiments, the oligonucleotidemay include at least one (G)m sequence, wherein m is 4 to 10. In somecases, at least one but up to all CG dinucleotides are unmethylated.According to some embodiments, the oligonucleotide may additionallycomprise a non-nucleotidic modification. The non-nucleotidicmodifications include but are not limited to: C₆-C₄₈-polyethyleneglycol,C₃-C₂₀-alkane-diol, C₃-C₁₈-alkylamino linker, C₃-C₁₈-alkylthiol linker,cholesterol, bile acid, saturated or unsaturated fatty acid, folate, ahexadecyl-glycerol or dihexadecyl-glycerol group, an octadecyl-glycerolor dioctadecyl-glycerol group, a vitamin E group. In other embodiments,the oligonucleotide of the invention further comprises a non-nucleotidicbrancher moiety or a nucleotidic brancher moiety. In some embodiments,the oligonucleotide includes a brancher moiety, wherein theoligonucleotides has at least two 5′-ends.

According to the invention, some embodiments include at least twonucleotides of the oligonucleotide have a stabilized linkage, including:phosphorothioate, phosphorodithioate, methylphosphonate,methylphosphonothioate boranophosphonate, phosphoramidate, or adephospho linkage, either as enantiomeric mixture or as enantiomericpure S- or R-configuration.

Yet in some embodiments, the YZ of R₁YZR₂ has a phosphodiester linkageor a phosphorothioate linkage. In some cases, the R₁Y and or the ZR₂ ofR₁YZR₂ has a phosphorothioate linkage. In some embodiments, all othernucleotides have a phosphorothioate linkage.

According to some embodiments of the invention, the oligonucleotide isfree of a microcarrier, including a lipid carrier.

According to the invention, the oligonucleotides may be an A classoligonucleotide, a B class oligonucleotide, a C class oligonucleotide, aP class oligonucleotide or a T class oligonucleotide. For the B classoligonucleotide of the invention, some embodiments include the sequence5′ TCN₁TX₁X₂CGX₃X₄ 3′, wherein X₁ is G or A; X₂ is T, G, or A; X₃ is Tor C and X₄ is T or C; and N is any nucleotide, and N₁ and N₂ arenucleic acid sequences of about 0-25 N's each.

According to some embodiments of the invention, the oligonucleotidecomprises at least one 3′-3′ linkage and or at least one 5′-5′ linkage.

In another aspect the invention is a composition of the oligonucleotidesdescribed herein in combination with an antigen or other therapeuticcompound, such as an anti-microbial agent. The anti-microbial agent maybe, for instance, an anti-viral agent, an anti-parasitic agent, ananti-bacterial agent or an anti-fungal agent.

A composition of a sustained release device including theoligonucleotides described herein is provided according to anotheraspect of the invention.

The composition may optionally include a pharmaceutical carrier and/orbe formulated in a delivery device. In some embodiments the deliverydevice is selected from the group consisting of cationic lipids, cellpermeating proteins, and sustained release devices. In one embodimentthe sustained release device is a biodegradable polymer or amicroparticle.

According to another aspect of the invention a method of stimulating animmune response is provided. The method involves administering anoligonucleotide to a subject in an amount effective to induce an immuneresponse in the subject. Preferably the oligonucleotide is administeredorally, locally, in a sustained release device, mucosally, systemically,parenterally, or intramuscularly. When the oligonucleotide isadministered to the mucosal surface it may be delivered in an amounteffective for inducing a mucosal immune response or a systemic immuneresponse. In preferred embodiments the mucosal surface is selected fromthe group consisting of an oral, nasal, rectal, vaginal, and ocularsurface.

In some embodiments the method includes exposing the subject to anantigen wherein the immune response is an antigen-specific immuneresponse. In some embodiments the antigen is selected from the groupconsisting of a tumor antigen, a viral antigen, a bacterial antigen, aparasitic antigen and a peptide antigen.

The oligonucleotides are useful for treating cancer in a subject havingcancer or in a subject at risk of developing a cancer (e.g., reducing arisk of developing cancer). The cancer may be selected from the groupconsisting of biliary tract cancer, breast cancer, cervical cancer,choriocarcinoma, colon cancer, endometrial cancer, gastric cancer,intraepithelial neoplasms, lymphomas, liver cancer, lung cancer (e.g.small cell and non-small cell), melanoma, neuroblastomas, oral cancer,ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer,sarcomas, thyroid cancer, and renal cancer, as well as other carcinomasand sarcomas. In some important embodiments, the cancer is selected fromthe group consisting of bone cancer, brain and CNS cancer, connectivetissue cancer, esophageal cancer, eye cancer, Hodgkin's lymphoma, larynxcancer, oral cavity cancer, skin cancer, and testicular cancer.

The oligonucleotides may also be used for increasing the responsivenessof a cancer cell to a cancer therapy (e.g., an anti-cancer therapy),optionally when the CpG immunostimulatory oligonucleotide isadministered in conjunction with an anti-cancer therapy. The anti-cancertherapy may be a chemotherapy, a vaccine (e.g., an in vitro primeddendritic cell vaccine or a cancer antigen vaccine) or an antibody basedtherapy. This latter therapy may also involve administering an antibodyspecific for a cell surface antigen of for example, a cancer cell,wherein the immune response results in antibody dependent cellularcytotoxicity (ADCC). In one embodiment, the antibody may be selectedfrom the group consisting of Ributaxin, Herceptin, Quadramet, Panorex,IDEC-Y2B8, BEC2, C225, Oncolym, SMART M195, ATRAGEN, Ovarex, Bexxar,LDP-03, ior t6, MDX-210, MDX-11, MDX-22, OV103, 3622W94, anti-VEGF,Zenapax, MDX-220. MDX-447, MELIMMUNE-2, MELIMMUNE-1, CEACIDE, Pretarget,NovoMAb-G2, TNT, Gliomab-H, GNI-250, EMD-72000, LymphoCide, CMA 676,Monopharm-C, 4B5, ior egf.r3, ior c5, BABS, anti-FLK-2, MDX-260, ANA Ab,SMART 1D10 Ab, SMART ABL 364 Ab and ImmuRAIT-CEA.

Thus, according to some aspects of the invention, a subject havingcancer or at risk of having a cancer is administered an oligonucleotideand an anti-cancer therapy. In some embodiments, the anti-cancer therapyis selected from the group consisting of a chemotherapeutic agent, animmunotherapeutic agent and a cancer vaccine.

The invention in other aspects relates to methods for preventing diseasein a subject. The method involves administering to the subject anoligonucleotide on a regular basis to promote immune systemresponsiveness to prevent disease in the subject. Examples of diseasesor conditions sought to be prevented using the prophylactic methods ofthe invention include microbial infections (e.g., sexually transmitteddiseases) and anaphylactic shock from food allergies.

In other aspects, the invention is a method for inducing an innateimmune response by administering to the subject an oligonucleotide in anamount effective for activating an innate immune response.

According to another aspect of the invention a method for treating aviral or retroviral infection is provided. The method involvesadministering to a subject having or at risk of having a viral orretroviral infection, an effective amount for treating the viral orretroviral infection of any of the compositions of the invention. Insome embodiments the virus is caused by a hepatitis virus e.g.,hepatitis B, hepatitis C, HIV, herpes virus, or papillomavirus.

A method for treating a bacterial infection is provided according toanother aspect of the invention. The method involves administering to asubject having or at risk of having a bacterial infection, an effectiveamount for treating the bacterial infection of any of the compositionsof the invention. In one embodiment the bacterial infection is due to anintracellular bacteria.

In another aspect the invention is a method for treating a parasiteinfection by administering to a subject having or at risk of having aparasite infection, an effective amount for treating the parasiteinfection of any of the compositions of the invention. In one embodimentthe parasite infection is due to an intracellular parasite. In anotherembodiment the parasite infection is due to a non-helminthic parasite.

In some embodiments the subject is a human and in other embodiments thesubject is a non-human vertebrate selected from the group consisting ofa dog, cat, horse, cow, pig, turkey, goat, fish, monkey, chicken, rat,mouse, and sheep.

In another aspect, the invention relates to a method for treatingautoimmune disease by administering to a subject having or at risk ofhaving an autoimmune disease an effective amount for treating orpreventing the autoimmune disease of any of the compositions of theinvention.

The invention in some aspects is a method for treating airwayremodeling, asthma or allergy comprising: administering to a subject anyof the compositions of the invention, in an effective amount to treatairway remodeling asthma or allergy in the subject. In one embodimentthe subject has asthma, chronic obstructive pulmonary disease, or is asmoker. In other embodiments the subject is free of symptoms of asthma.

Use of an oligonucleotide of the invention for stimulating an immuneresponse is also provided as an aspect of the invention.

A method for manufacturing a medicament of an oligonucleotide of theinvention for stimulating an immune response is also provided.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention. This invention is not limited in its application to thedetails of construction and the arrangement of components set forth inthe following description or illustrated in the drawings. The inventionis capable of other embodiments and of being practiced or of beingcarried out in various ways. Also, the phraseology and terminology usedherein is for the purpose of description and should not be regarded aslimiting. The use of “including,” “comprising,” or “having,”“containing”, “involving”, and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are two drawings illustrating the structure of themodified bases of the invention. FIG. 1A shows a section of a CpGhexamer motif (GTCGTT). FIG. 1B shows the incorporated hydrophobic shapeanalogs of 2′-deoxythymidine: 2,4-Difluorotoluene (FF), 5-bromouridine(BU) and 5-iodouridine (JU).

FIG. 2 is a graph showing results of a luciferase assay with B-classoligonucleotides (ODN) modified with thymine shape analog2,4-difluorotoluene (FF). The activity of FF-modified ODN (SEQ IDNO:3-9) was compared to that of the unmodified B-class parent sequence(SEQ ID NO:1), fully PS parent sequence (SEQ ID NO:2), and a thirdunmodified B-class ODN (SEQ ID NO:37). hTLR9-LUC-293 cells werestimulated with indicated amounts of ODN and NF-kB stimulation wasdetermined by measuring luciferase activity 16 h later. The x-axis islog ODN concentration in μM and the y-axis is the relative stimulationindex.

FIG. 3 is a graph demonstrating the results of a luciferase assay withmodified B-class ODN. Thymidine (T) was substituted with5-bromo-2′-deoxyuridine (BU) (SEQ ID NO:10-12) and5-iodo-2′-deoxyuridine (JU) (SEQ ID NO:13-15). Their activity wascompared to that of the unmodified B-class parent sequence (SEQ IDNO:1), fully PS parent sequence (SEQ ID NO:2), and a third unmodifiedB-class ODN (SEQ ID NO:37). hTLR9-LUC-293 cells were stimulated withindicated amounts of ODN and NF-κB stimulation was determined bymeasuring Luciferase activity 16 h later. The x-axis is log ODNconcentration in μM and the y-axis is the relative stimulation index.

FIG. 4 is a graph demonstrating the results of a luciferase assay withmodified B-class ODN. 2′-deoxythymidine (T) was substituted with2′-deoxyuridine (U) (SEQ ID NO:16-18). The activity of the U-modifiedODN was compared to that of the unmodified B-class parent sequence (SEQID NO:1), fully PS parent sequence (SEQ ID NO:2), and a third unmodifiedB-class ODN (SEQ ID NO:37). hTLR9-LUC-293 cells were stimulated withindicated amounts of ODN and NF-κB stimulation was determined bymeasuring Luciferase activity 16 h later. The x-axis is log ODNconcentration in μM and the y-axis is the relative stimulation index.

FIGS. 5a-b are two graphs demonstrating the results of a luciferaseassay and a PBMC assay with modified B class ODN. The relative activityof an ODN with 5-Ethyl- 2′-deoxyuridine (EU) (SEQ ID NO:42),2′-deoxyuridine (U) (SEQ ID NO:16), 5-iodo-2′- deoxyuridine (JU) (SEQ IDNO:13), 5-bromo-2′-deoxyuridine (BU) (SEQ ID NO:10), and5-Chloro-2′-deoxyuridine (CU) (SEQ ID NO:41) was compared to that of theparent sequence (SEQ ID NO:1). FIG. 5a shows TLR9 activity and FIG. 5bshows IFN- alpha production. Shown is the mean+/−SEM of three donors.The x-axes are ODN concentration in μM and the y-axes are the relativestimulation index (FIG. 5a ) or IFN- alpha concentration in μg/ml (FIG.5b ).

FIG. 6 is a graph demonstrating the results of a luciferase assay withEU-modified ODN. The activity of EU-modified ODN SEQ ID NO:29, 30, and42 was compared to that of the parent sequence (SEQ ID NO:1) and anotherunmodified B-class ODN (SEQ ID NO:37). The x-axis is ODN concentrationin μM and the y-axis is the relative stimulation index.

FIG. 7 is a graph demonstrating the results of a luciferase assay withmodified B class ODN. The activity of JU-modified SEQ ID NO:19-24 wascompared to that of parent sequence SEQ ID NO:37. The x-axis is ODNconcentration in μM and the y-axis is the relative stimulation index.

FIGS. 8a-b are two graphs demonstrating the results of a luciferaseassay and a PBMC assay with modified A class ODN. The activity ofJU-modified SEQ ID NO:35- 37 was compared to that of the unmodifiedparent sequence (SEQ ID NO:43) and to unmodified B-class ODN SEQ IDNO:1. FIG. 8a shows TLR9 activity and FIG. 8b shows IFN-alphaproduction. Shown is the mean +/−SEM of three donors. The x-axes are logODN concentration (FIG. 8a ) or ODN concentration (FIG. 8b ) in μM andthe y-axes are the relative stimulation index index (FIG. 8a ) orIFN-alpha concentration in pg/ml (FIG. 8b ).

FIG. 9 is a graph demonstrating the results of a luciferase assay withmodified C class ODN. The activity of JU-modified C-class ODN SEQ IDNO:27-28 and 44-45 was compared to that of the unmodified parentsequence SEQ ID NO:45 and to an unmodified B-class ODN (SEQ ID NO:37).The x-axis is ODN concentration in μM and the y-axis is the relativestimulation index.

FIG. 10 is a graph demonstrating the results of a luciferase assay withmodified P class ODN. The activity of JU-modified SEQ ID NO:31-33 wascompared to that of the unmodified parent sequence (SEQ ID NO:52). Thex-axis is log ODN concentration in μM and the y-axis is the relativestimulation index.

FIG. 11 is a graph demonstrating the results of a luciferase assay withmodified T class ODN. The activity of JU-modified SEQ ID NO:47-50 andU-modified SEQ ID NO:51 was compared to that of unmodified parentsequence SEQ ID NO:25. The x-axis is log ODN concentration in μM and they-axis is the relative stimulation index.

FIGS. 12a-b are graphs demonstrating the results of a luciferase assaywith short ODN. The activity of JU-modified short ODN SEQ ID NO:39-40was compared to that of the unmodified parent sequence SEQ ID NO:38 andto the B-class ODN SEQ ID NO:37. ODN were formulated with and withoutDOTAP. The x-axis is log ODN concentration in μM and the y-axis is therelative stimulation index.

FIGS. 13a-d are four graphs showing the results of an ELISA assaymeasuring cytokine concentration in in splenocyte culture supernatantswhere BALB/c mouse splenocytes were cultured with different ODNs.Culture supernatants were harvested at 6 hr (for TNF-alpha) or 24 hr(for IL-6, IL-10 and IL-12). The activities of a JU- modified B-classODN (SEQ ID NO:13), an unmodified B-class ODN (SEQ ID NO:37), and anon-CpG negative control ODN (SEQ ID NO:26) were compared. FIGS. 13a-dshow TNF-alpha, IL-6, IL-10, and IL-12 concentration, respectively. Thex-axes are ODN concentration in μg/ml and the y-axes are cytokineconcentration in pg/ml.

FIG. 14 is a graph showing the results of FACS analysis of B cellproliferation. CPSE stained BALB/c mouse splenocytes (4×10⁵/well) wereincubated with 0.001, 0.01, 0.1, 0.3, 1, 3 or 10 μg/ml of ODN. At 72hours post incubation, cells were stained for CD19 and B-cellproliferation was determined by FACS followed by analysis by ModFitSoftware. The activities of a JU-modified B-class ODN (SEQ ID NO:13), anunmodified B-class ODN (SEQ ID NO:37), and a non-CpG negative controlODN (SEQ ID NO:26) were compared. The x-axis is ODN concentration inμg/ml and the y-axis is relative B cell proliferation.

FIGS. 15a-b are two graphs showing in vivo cytokine production asmeasured by ELISA. BALB/c mice (5 per group) were injected SC with 10,50 or 100μg of ODN. Control group received 100μl of PBS alone. Animalswere bled by cardiac puncture at 1 hour (for TNF-alpha) or 3 hour (forIP-10) post injection and plasma assayed for TNF-alpha and IP-10 byELISA. The activities of a JU-modified B-class ODN (SEQ ID NO:13) and anunmodified B-class ODN (SEQ ID NO:37) were compared. FIG. 15a showsTNF-alpha concentration and FIG. 15b shows IP-10 concentration. Thex-axes are ODN dose in μg and the y-axes are cytokine concentration inpg/ml.

FIG. 16 is a graph showing TLR9-mediated NF-κB activation by a B-classODN with a universal base (6-nitrobenzimidazol) (SEQ ID NO:178) in placeof thymidine in the parent sequence (SEQ ID NO:1). hTLR9-LUC-293 cellswere incubated with indicated amounts of nucleic acids and NF-κBactivation was determined 16 h later by measuring luciferase activity.The x-axis is log of ODN concentration in μM and the y-axis is IFN-αconcentration in pg/ml.

FIG. 17 is a graph showing TLR9-mediated NF-κB activation by B-class ODNwith 5-(2-bromovinyl)-uridine (SEQ ID NO:153 and 154) in place ofthymine in the parent sequence (SEQ ID NO:1). hTLR9-LUC-293 cells wereincubated with indicated amounts of nucleic acids and NF-κB activationwas determined 16 h later by measuring luciferase activity. The x-axisis log of ODN concentration in μM and the y-axis is IFN-α concentrationin pg/ml.

FIG. 18 is a graph showing TLR9-mediated NF-κB activation by B-class ODNwith a sugar modification (2′-O-methylguanosine) in addition to alipophilic substituted nucleotide analog (SEQ ID NO:111-113). Theactivity of these ODN was compared to that of the parent sequence (SEQID NO:1) and the same sequence with a lipophilic substituted nucleotideanalog only (SEQ ID NO:13). hTLR9-LUC-293 cells were incubated withindicated amounts of nucleic acids and NF-κB activation was determined16 h later by measuring luciferase activity. The x-axis is log of ODNconcentration in μM and the y-axis is IFN-α concentration in pg/ml.

FIG. 19 is a graph showing TLR9-mediated NF-κB activation by ranchedB-class ODN with multiple 5′ accessible ends. The activity of thebranched ODN (SEQ ID NO:96, 97, 101, and 102) was compared to that ofSEQ ID NO:1. hTLR9-LUC-293 cells were incubated with indicated amountsof nucleic acids and NF-κB activation was determined 16 h later bymeasuring luciferase activity. The x-axis is log of ODN concentration inμM and the y-axis is IFN-α concentration in pg/ml.

FIG. 20 is a graph showing TLR9-mediated NF-κB activation by a shortunmodified B-class ODN (SEQ ID NO:38) and an ODN of the same sequencewith a lipophilic substituted nucleotide analog and a lipophilic 3′ tag(SEQ ID NO:126). Both were formulated with and without DOTAP.hTLR9-LUC-293 cells were incubated with indicated amounts of nucleicacids and NF-κB activation was determined 16 h later by measuringluciferase activity. The x-axis is log of ODN concentration in μM andthe y-axis is IFN-α concentration in pg/ml.

FIG. 21 is a graph showing TLR9-mediated NF-κB activation by two B-classODN with 5-proynyl-dU (SEQ ID NO:116 and 117) in place of thymine of theparent sequence (SEQ ID NO:1). hTLR9-LUC-293 cells were incubated withindicated amounts of nucleic acids and NF-κB activation was determined16 h later by measuring luciferase activity. The x-axis is log of ODNconcentration in μM and the y-axis is IFN-α concentration in pg/ml.

FIG. 22 is a graph showing hTLR9-mediated NF-κB activation by B-classODN with a second nucleotide analog in addition to a lipophilicsubstituted nucleotide analog (SEQ ID NO:138, 7-deaza-dG; SEQ ID NO:139,inosine; SEQ ID NO:140, 5-methyl-dC). The activity of these ODN wascompared to that of the parent sequence (SEQ ID NO:1) and the samesequence with a lipophilic substituted nucleotide analog only (SEQ IDNO:13). hTLR9-LUC-293 cells were incubated with indicated amounts ofnucleic acids and NF-κB activation was determined 16 h later bymeasuring luciferase activity. The x-axis is log of ODN concentration inμM and the y-axis is IFN-α concentration in pg/ml.

FIG. 23 is a graph showing hTLR9-mediated NF-κB activation by T-classODN with a lipophilic substituted nucleotide analog (SEQ ID NO:132-134).The activity of these was compared to that of an immunostimulatoryC-class ODN (SEQ ID NO:198). hTLR9-LUC-293 cells were incubated withindicated amounts of nucleic acids and NF-κB activation was determined16 h later by measuring luciferase activity. The x-axis is log of ODNconcentration in μM and the y-axis is IFN-α concentration in pg/ml.

FIGS. 24a-b are two graphs showing hTLR9-mediated NF-KB activation byP-class 10 ODN with a lipophilic substituted nucleotide analog (SEQ IDNO:58-63). FIG. 24a shows the activity of SEQ ID NO:58-61 compared tothat of a B-class positive control (SEQ ID NO:55) and an unmodifiedP-class ODN (SEQ ID NO:56). FIG. 24b shows the activity of SEQ IDNO:62-63compared to that of the same positive and negative controls.hTLR9-LUC-293cells were incubated with indicated amounts of nucleicacids and NF-KB activation was determined 16h later by measuringluciferase activity. The x-axis is log of ODN concentration in μM andthe y-axis is the relative stimulation index.

FIG. 25 is a graph showing hTLR9-mediated NF-κB activation by P-classODN with a lipophilic substituted nucleotide analog (SEQ ID NO:64,66-67). The activity of these is compared to that of a B-class positivecontrol (SEQ ID NO:55), a C-class ODN (SEQ ID NO:68) and an unmodifiedP-class ODN (SEQ ID NO:57). hTLR9-LUC-293 cells were incubated withindicated amounts of nucleic acids and NF-κB activation was determined16 h later by measuring luciferase activity. The x-axis is log of ODNconcentration in μM and the y-axis is the relative stimulation index.

FIGS. 26a-b are two graphs showing induction of IFN-αby P-class ODN witha lipophilic substituted nucleotide analog (SEQ ID NO:58-63). FIG. 26ashows the activity of SEQ ID NO:58-61 compared to that of a B-classpositive control (SEQ ID NO:55) and an unmodified P-class ODN (SEQ IDNO:56). FIG. 26b shows the activity of SEQ ID NO:62-63 compared to thatof the same positive and negative controls. Human PBMC were incubatedwith the indicated ODN for 48 hours. IFN- αwas then determined in thecell culture supernatants by ELISA. The x-axes are ODN concentration inμM and the y-axes are IFN-αconcentration in pg/ml.

FIG. 27 is a graph showing induction of IFN-α by P-class ODN with alipophilic substituted nucleotide analog (SEQ ID NO:64, 66-67). Theactivity of these is compared to that of a B-class positive control (SEQID NO:55), a C-class ODN (SEQ ID NO:68) and an unmodified P-class ODN(SEQ ID NO:57). Human PBMC were incubated with the indicated ODN for 48hours. IFN-α was then determined in the cell culture supernatants byELISA. The x-axes are ODN concentration in μM and the y-axes are IFN-αconcentration in pg/ml.

FIGS. 28a-b are two graphs showing IL-6 induction by P-class ODN with alipophilic substituted nucleotide analog (SEQ ID NO:58, 60-62, FIG. 28a) (SEQ ID NO:64 and 67, FIG. 28b ). The activity was compared to that ofan unmodified B-class ODN (SEQ ID NO:55), and unmodified C-class ODN(SEQ ID NO:54), a negative control ODN (SEQ ID NO:53), and an unmodifiedP-class ODN (SEQ ID NO:56). PBMC from three donors were incubated withthe ODN for 24 hours and the supernatants were analyzed by luminex.Shown is the mean +/−SEM. The x-axes are ODN concentration in μM and 15the y-axes are IL-6concentration in pg/ml.

FIGS. 29a-b are two graphs showing B-cell proliferation after treatmentwith P-class class ODN with a lipophilic substituted nucleotide analog(SEQ ID NO:58, 60-62, FIG. 29a ) (SEQ ID NO:64 and 67, FIG. 29b ). Theactivity was compared to that of an unmodified B-class ODN (SEQ IDNO:55), an unmodified C-class ODN (SEQ ID 20 NO:54), a negative controlODN (SEQ ID NO:53), an unmodified P-class ODN (SEQ ID NO:56), LPS,R-848, SEB, and a poly[I]:[C] ODN. CFSE-labeled PBMC from three donorswere incubated with the ODN for 5 days and then stained with a CD19antibody. The percentage of B cells with reduced CFSE staining wasdetermined. The x-axes are ODN concentration in μM and the y-axes are %of B cells with reduced staining after division.

FIG. 30 is a graph showing induction of murine IFN-α by P-class ODN witha lipophilic substituted nucleotide analog (SEQ ID NO:58, 60-62, 64, and67). The activity of these is compared to that of a B-class positivecontrol (SEQ ID NO:55) and a negative control (SEQ ID NO:26). BALB/cmice (5 per group) were injected SC with differing doses of ODN. Animalswere bled at 3 hr post injection and plasma tested for IFN-alpha byELISA. The x axis is ODN dose in μg and the y-axis is IFN-αconcentration in pg/ml.

FIGS. 31a-b are two graphs showing the effect of ODN on tumor volume inthe mouse SA1N tumor model. Female A/J mice (10 per group) were injectedSC with 5 ×10⁵ Sal/N tumor cells on day 0. Mice were treated with35μ(FIG. 31a ) or 100μg (FIG. 31b ) P-class ODN with a lipophilicsubstituted nucleotide analog (SEQ ID NO:60, 64, and 67), an unmodifiedC-class ODN, an unmodified B-class ODN (SEQ ID NO:55), or PBS alonegiven SC once weekly starting on day 8 post tumor induction. Animalswere monitored for survival and tumor volume. Tumor size (the length andthe width) was measured using a digital vernier caliper. Tumor volumewas calculated by using the formula: Tumor volume =(0.4) (ab2), where a=large diameter and b=smaller diameter. The x-axes show days post tumorinduction and the y-axes show tumor volume in mm³.

DETAILED DESCRIPTION

The invention is based in part on CpG oligonucleotides that showenhanced immunostimulatory capacity. CpG oligonucleotides are known tostimulate the immune system, for example through interaction withtoll-like receptor 9 (TLR9). Stimulation of TLR9 has many effectsincluding stimulation of a Th1 biased immune response, NK cellactivation and B cell activation. The invention is related in someaspects to the identification of immunostimulatory oligonucleotides withaltered structure that affects their interaction with TLR9. It wasdiscovered by the inventors that oligonucleotides with lipophilicsubstituted nucleotide analogs outside the CpG motif have enhancedability to stimulate interferon-α (IFN-α) production and induce TLR9activation. This effect has been observed in all classes ofimmunostimulatory oligonucleotides tested. These modifiedoligonucleotides with enhanced stimulatory capacity have been termed Bclass oligonucleotides.

The E class modified oligonucleotides of the instant invention have insome instances enhanced capacity for inducing an immune response. Aninduction of an immune response refers to any increase in number oractivity of an immune cell, or an increase in expression or absolutelevels of an immune factor, such as a cytokine. Immune cells include,but are not limited to, NK cells, CD4+ T lymphocytes, CD8+ Tlymphocytes, B cells, dendritic cells, macrophage and otherantigen-presenting cells. Cytokines include, but are not limited to,interleukins, TNF-α, IFN-α,β and γ, Flt-ligand, and co-stimulatorymolecules.

It is known that oligonucleotides containing unmethylated CpG motifs areable to stimulate immune responses through the Toll-like receptor 9(TLR9) pathway. The induction of many cytokines correlates with TLR9activation. Thus induction increases a TLR9 stimulation increases.However there is generally an inverse correlation between TLR9 and IFN-αinduction for CpG ODN. It was discovered that some of the modificationsof the invention can produce a modified signaling pattern such that amore direct correlation, rather than an inverse correlation between TLR9activation and IFN-α is observed.

The inventors set out to investigate the impact of the lipophilicresidues in region surrounding the CpG motif. As described in theexamples below several different types of lipophilic substitutednucleotide analogs, such as 2,4-difluorotoluene, 5-bromouracil and5-iodouracil were incorporated into a CpG oligonucleotide on either the5′ or 3′ side of the CpG motif Unexpectedly, incorporation of theselipophilic substituted nucleotide analogs led to an unusually strongincrease in hTLR9 activity as well as IFN-α induction in human PBMC'sSubstitution with a non-lipophilic nucleotide such as a uracil residue(which is structurally similar to a thymine but lacking a methyl group)produced a strong decrease in hTLR9 stimulation. In the oligonucleotidestested, the increase in TLR9 stimulation appeared to be better if thelipophilic substituted nucleotide analog is positioned 5′ to the CpGmotif than when it was positioned 3′ to the motif. Double substitution(i.e. a 5′ and 3′ lipophilic substituted nucleotide analog substitution)resulted in most potent stimulation of those tested. In contrast,substitution of guanin or cytosine by 2,4-difluorotoluene at the CpGmotif led in both cases to a strong decrease of the TLR9 stimulationindex.

The lipophilic substituted nucleotide analogs modification resulted in astrong enhancement of IFN-α induction. Especially, for the 5-bromouraciland 5-iodouracil modified ODN, there appeared to be a good correlationbetween TLR9 stimulation and IFN-α induction. As mentioned above, thisobservation was unexpected, since (i) the parent molecule 21317 isvirtually inactive in inducing IFN-α and (ii) there is usually aninverse correlation between TLR9 and IFN-α induction for CpG ODN whichdo not contain these modifications.

In some aspects of the invention the oligonucleotide has the sequenceR₁YZR₂. The olignucleotide may be include one or more such motifs. R₁and R₂ are independently any one of lipophilic substituted nucleotideanalog (L), a nucleotide, or a linkage. It is preferred, however, thatat least one of R₁ and R₂ is a lipophilic substituted nucleotide analog(L). In some instances R₁ and R₂ are both L. As shown in the examplessection below oligonucleotides having an L both 5′ and 3′ to the CpGmotif were particularly stimulatory. However sometimes only one R is anL. For instance R₁ may be L and R₂ is a nucleotide or vice versa.Alternatively R₁ may be a L and R₂ may be a linkage, such that theoligonucleotide comprises a structure 5′ R₁CG 3′.

In some instances the oligonucleotide has the sequence R₁N₁YZN₂R₂wherein N₁ and N₂ are nucleotides of 0-3 nucleotides in length. Otherpossible variations include structures such as 5′ R₁N₁R₁YZ N₂ 3′, 5′R₃R₁YZ 3 and R₁ZN₂R₂.

Y is a pyrimidine nucleotide. Z is a purine, a pyrimidine, or an abasicresidue. In some embodiments Z is preferably a purine.

L is a lipophilic substituted nucleotide analog which may be, forinstance, a 5- or 6-membered ring nucleobase analog. An example of a 5-or 6-membered ring nucleobase analog is shown in the following group offormula I.

A, B, X, D, E, and F are independently any one of C (carbon) or N(nitrogen) optionally bearing hydrogen or a substituent such as forinstance, but not limited to, F, Cl, Br, I, alkyl, alkenyl, alkinyl,halogenated alkyl, halogenated alkenyl, cycloalkyl, O-alkyl, O-alkenyl,—NH-alkyl, —N(alkyl)₂; —S-alkyl, —SO-alkyl, —SO₂-alkyl, nitro, cyano,carboxylester, phenyl, thiophenyl, benzyl, oxo, thio, hydroxy, mercapto,and imino. In some instances, at least one substituent is not oxo, thio,hydroxy, mercapto, imino, amino or methyl. n is 0 or 1. The dotted linesindicate optional double bonds. However, at least one substituent is notchosen from the group consisting of oxo, thio, hydroxy, mercapto, imino,amino, methyl and hydrogen. Additionally the total of A, B, X, D, B andP atoms is not more than 3 nitrogens (N). In some embodiments all atomsA, B, X, D, B, F are carbon (C). Alternatively, at least one, two, orthree of the atoms A, B, X, D, B, F is nitrogen (N).

The compound of formula may be, for example, any of the followinglipophilic substituted nucleotide analogs: a substituted pyrimidine, asubstituted uracil, a substituted toluene, a substituted imidazole orpyrazole, a substituted triazole, 5-chloro-uracil, 5-bromo-uracil,5-iodo-uracil, 5-ethyl-uracil, 5-propyl-uracil, 5-propinyl-uracil,(E)-5-(2-bromovinyl)-uracil, or 2.4-difluoro-toluene.

The lipophilic substituted nucleotide analog may be separate or it maybe fused with another compound. For instance is may be fused to a 3-to-6-membered aromatic or aliphatic ring system. It may also be linkedto a 5- to 6-membered sugar moiety such as for instance a pentose orhexose. An example of a pentose is a furanose such as a ribose ordeoxyribose and an example of a hexose is a pyranose. The pentose orhexose can optionally be substituted by F, amino, alkoxy, alkoxy-ethoxy,amonipropyl, alkenyl, alkinyl, or a O2,C4-alkylene bridge.

The oligonucleotide may also include a non-nucleotidic modification suchas a C₆-C₄₈-polyethyleneglycol, C₃-C₂₀-alkane-diol, C₃-C₁₈-alkylaminolinker, C₃-C₁₈-alkylthiol linker, cholesterol, bile acid, saturated orunsaturated fatty acid, folate, hexadecyl-glycerol, dihexadecyl-glycerolgroup, an octadecyl-glycerol or dioctadecyl-glycerol group or a vitaminE group.

The lipophilic substituted nucleotide analogs can be incorporated intoany immunostimulatory oligonucleotide. In some embodiments of theinvention the immunostimulatory oligonucleotides includeimmunostimulatory motifs which are “CpG dinucleotides”. A CpGdinucleotide can be methylated or unmethylated. An immunostimulatorynucleic acid containing at least one unmethylated CpG dinucleotide is anucleic acid molecule which contains an unmethylated cytosine-guaninedinucleotide sequence (i.e., an unmethylated 5′ cytidine followed by 3′guanosine and linked by a phosphate bond) and which activates the immunesystem; such an immunostimulatory nucleic acid is a CpG nucleic acid.CpG nucleic acids have been described in a number of issued patents,published patent applications, and other publications, including U.S.Pat. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; and6,339,068. An immunostimulatory nucleic acid containing at least onemethylated CpG dinucleotide is a nucleic acid which contains amethylated cytosine-guanine dinucleotide sequence (i.e., a methylated 5′cytidine followed by a 3′ guanosine and linked by a phosphate bond) andwhich activates the immune system. In other embodiments theimmunostimulatory oligonucleotides are free of CpG dinucleotides. Theseoligonucleotides which are free of CpG dinucleotides we referred to asnon-CpG oligonucleotides, and they have non-CpG immunostimulatorymotifs. Preferably these are T-rich ODN, such as ODN having at least 80%T.

The E class ODNs of the invention may include motifs and properties ofother CpG ODN classes such as A class, B call, C class, T class and Pclass as long as they include lipophilic substituted nucleotide analogs5′ and/or 3′ of a YGZ motif.

“A class” CpG immunostimulatory nucleic acids have been described inU.S. Non-Provisional patent application Ser. No. 09/672,126 andpublished PCT application PCT/US00/26527 (WO 01/22990), both filed onSep. 27, 2000. These nucleic acids are characterized by the ability toinduce high levels of interferon-alpha while having minimal effects on Bcell activation. The A class CpG immunostimulatory nucleic acid do notnecessarily contain a hexamer palindrome GACGTC, AGCGCT, or AACGTTdescribed by Yamamoto and colleagues. Yamamoto S et al. J Immunol148:4072-6 (1992).

Exemplary sequences of A class immunostimulatory nucleic acids aredescribed in U.S. Non-Provisional patent application Ser. No. 09/672,126and published PCT application PCT/US00/26527 (WO 01/22990), both filedon Sep. 27, 2000.

“B class” ODN are potent at activating B cells but we relatively weak ininducing IFN-α and NK cell activation. The B class CpG nucleic acidstypically are fully stabilized and include an unmethylated CpGdinucleotide within certain preferred base contexts. See, e.g., U.S.Pat. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; and6,339,068. Another class is potent for inducing IFN-α and NK cellactivation but is relatively weak at stimulating B cells; this class hasbeen termed the “A class”. The A class CpG nucleic acids typically havestabilized poly-G sequences at 5′ and 3′ ends and a palindromicphosphodiester CpG dinucleotide-containing sequence of at least 6nucleotides. See, for example, published patent applicationPCT/US00/26527

Yet another class of CpG nucleic acids activates B cells and NK cellsand induces IFN-α; this class has been termed the C-class. The “C class”immunostimulatory nucleic acids contain at least two distinct motifshave unique and desirable stimulatory effects on cells of the immunesystem. Some of these ODN have both a traditional “stimulatory” CpGsequence and a “GC-rich” or “B-cell neutralizing” motif. Thesecombination motif nucleic acids have immune stimulating effects thatfall somewhere between those effects associated with traditional “classB” CpG ODN, which are strong inducers of B cell activation and dendriticcell (DC) activation, and those effects associated with a more recentlydescribed class of immune stimulatory nucleic acids (“class A” CpG ODN)which are strong inducers of IFN-α and natural killer (NK) cellactivation but relatively poor inducers of B-cell and DC activation.Krieg A M et al. (1995) Nature 374:546-9; Ballas Z K et al. (1996) JImmunol 157:1840-5; Yamamoto S et al. (1992) J Immunol 148:4072-6. Whilepreferred class B CpG ODN often have phosphorothioate backbones andpreferred class A CpG ODN have mixed or chimeric backbones, the C classof combination motif immune stimulatory nucleic acids may have eitherstabilized, e.g., phosphorothioate, chimeric, or phosphodiesterbackbones, and in some preferred embodiments, they have semi-softbackbones. This class has been described in U.S. patent application U.S.Ser. No. 10/224,523 filed on Aug. 19, 2002, the entire contents of whichis incorporated herein by reference.

The “P class” immunostimulatory oligonucleotides have several domains,including a 5′TLR activation domain, 2 duplex forming regions and anoptional spacer and 3′ tail. This class of oligonucleotides has theability in some instances to induce much higher levels of IFN-αsecretion than the C-Class. The P-Class oligonucleotides have theability to spontaneously self-assemble into concatamers either in vitroand/or in vivo. Without being bound by any particular theory for themethod of action of these molecules, one potential hypothesis is thatthis property endows the P-Class oligonucleotides with the ability tomore highly crosslink TLR9 inside certain immune cells, inducing adistinct pattern of immune activation compared to the previouslydescribed classes of CpG oligonucleotides. Cross-linking of TLR9receptors may induce activation of stronger IFN-α secretion through thetype I IFNR feedback loop in plasmacytoid dendritic cells. P classoligonucleotides are described at least in U.S. application Ser. No.11/706,561.

The “T class” oligonucleotides induce secretion of lower levels ofIFN-alpha when not modified as in the ODNs of the invention andIFN-related cytokines and chemokines than B class or C classoligonucleotides, while retaining the ability to induce levels of IL-10similar to B class oligonucleotides. T class oligonucleotides aredescribed at least in U.S. patent application Ser. No. 11/099,683, theentire contents of which are hereby incorporated by reference.

In one embodiment the immunostimulatory ODN of the invention isadvantageously combined with a cationic lipid. In one embodiment thecationic lipid is DOTAP(N-[1-(2,3-dioleoyloxy)propy-1]-N,N,N-trimethylammonium methyl-sulfate).Other agents with similar properties including trafficking to theendosomal compartment can be used in place of or in addition to DOTAP.Other lipid formulations include, for example, as EFFECTENE™ (anon-liposomal lipid with a special DNA condensing enhancer) andSUPERFECT™ (a novel acting dendrimeric technology). Liposomes arecommercially available from Gibco BRL, for example, as LIPOFECTIN™ andLIPOFECTACE™, which are formed of cationic lipids such as N-[1-(2,3dioleyloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA) anddimethyl dioctadecylammonium bromide (DDAB). Methods for makingliposomes are well known in the art and have been described in manypublications. Liposomes also have been reviewed by Gregoriadis G (1985)Trends Biotechnol 3:235-241.

In other embodiments the immunostimulatory ODN are not formulated incationic liposomes. Due to the lipophilic nature of the modified analogswithin the ODN even short ODN such as 3 nucleotides in length may notrequire formulation to efficiently function in vivo.

In one embodiment the immunostimulatory ODN of the invention are in theform of covalently closed, dumbbell-shaped molecules with both primaryand secondary structure. In one embodiment such cyclicoligoribonucleotides include two single-stranded loops connected by anintervening double-stranded segment. In one embodiment at least onesingle-stranded loop includes an immunostimulatory DNA motif of theinvention. Other covalently closed, dumbbell-shaped molecules of theinvention include chimeric DNA:RNA molecules in which, for example, thedouble-stranded segment is at least partially DNA (e.g., eitherhomodimeric dsDNA or heterodimeric DNA:RNA) and at least onesingle-stranded loop includes an immunostimulatory DNA motif of theinvention. Alternatively, the double stranded segment of the chimericmolecule is DNA.

In certain embodiments the immunostimulatory ODN is isolated. Anisolated molecule is a molecule that is substantially pure and is freeof other substances with which it is ordinarily found in nature or in invivo systems to an extent practical and appropriate for its intendeduse. In particular, the immunostimulatory ODN are sufficiently pure andare sufficiently free from other biological constituents of cells so asto be useful in, for example, producing pharmaceutical preparations.Because an isolated immunostimulatory ODN of the invention may beadmixed with a pharmaceutically acceptable carrier in a pharmaceuticalpreparation, the immunostimulatory ODN may comprise only a smallpercentage by weight of the preparation. The immunostimulatory ODN isnonetheless substantially pure in that it has been substantiallyseparated from the substances with which it may be associated in livingsystems.

The immunostimulatory nucleic acid molecules may have a chimericbackbone. For purposes of the instant invention, a chimeric backbonerefers to a partially stabilized backbone, wherein at least oneinternucleotide linkage is phosphodiester or phosphodiester-like, andwherein at least one other internucleotide linkage is a stabilizedinternucleotide linkage, wherein the at least one phosphodiester orphosphodiester-like linkage and the at least one stabilized linkage aredifferent. Since boranophosphonate linkages have been reported to bestabilized relative to phosphodiester linkages, for purposes of thechimeric nature of the backbone, boranophosphonate linkages can beclassified either as phosphodiester-like or as stabilized, depending onthe context. For example, a chimaeric backbone according to the instantinvention could in one embodiment include at least one phosphodiester(phosphodiester or phosphodiester-like) linkage and at least oneboranophosphonate (stabilized) linkage. In another embodiment a chimericbackbone according to the instant invention could includeboranophosphonate (phosphodiester or phosphodiester-like) andphosphorothioate (stabilized) linkages. A “stabilized internucleotidelinkage” shall mean an internucleotide linkage that is relativelyresistant to in vivo degradation (e.g., via an exo- or endo-nuclease),compared to a phosphodiester internucleotide linkage. Preferredstabilized internucleotide linkages include, without limitation,phosphorothioate, phosphorodithioate, methylphosphonate, andmethylphosphorothioate. Other stabilized internucleotide linkagesinclude, without limitation: peptide, alkyl, dephospho, and others asdescribed above.

Modified backbones such as phosphorothioates may be synthesized usingautomated techniques employing either phosphoramidate or H-phosphonatechemistries. Aryl- and alkyl-phosphonates can be made, e.g., asdescribed in U.S. Pat. No. 4,469,863; and alkylphosphotriesters (inwhich the charged oxygen moiety is alkylated as described in U.S. Pat.No. 5,023,243 and European Patent No. 092,574) can be prepared byautomated solid phase synthesis using commercially available reagents.Methods for making other DNA backbone modifications and substitutionshave been described. Uhlmann E et al (1990) Chem Rev 90:544; Goodchild J(1990) Bioconjugate Chem 1:165. Methods for preparing chimericoligonucleotides are also known. For instance patents issued to Uhlmannet al have described such techniques.

Mixed backbone modified ODN may be synthesized using a commerciallyavailable DNA synthesizer and standard phosphoramidite chemistry. (P. B.Eckstein, “Oligonucleotides and Analogs—A Practical Approach” IRL Press,Oxford, UK, 1991, and M. D. Matteucci and M. H. Caruthers, TetrahedronLett. 21, 719 (1980)) After coupling, PS linkages are introduced bysulfurization using the Beaucage reagent (R. P. Iyer, W. Egan. J. B.Regan and S. L. Beaucage, J. Am. Chem. Soc. 112, 1253 (1990)) (0.075 Min acetonitrile) or phenyl acetyl disulfide (PADS) followed by cappingwith acetic anhydride, 2,6-lutidine in tetrahydrofurane (1:1:8; v:v:v)and N-methylimidazole (16% in tetrahydrofurane). This capping step isperformed after the sulfurization reaction to minimize formation ofundesired phosphodiester (PO) linkages at positions where aphosphorothioate linkage should be located. In the case of theintroduction of a phosphodiester linkage, e.g. at a CpG dinucleotide,the intermediate phosphorous-III is oxidized by treatment with asolution of iodine in water/pyridine. After cleavage from the solidsupport and final deprotection by treatment with concentrated ammonia(15 hrs at 50° C.), the ODN are analyzed by HPLC on a Gen-Pak Fax column(Millipore-Waters) using a NaCl-gradient (e.g. buffer A: 10 mM NaH₂PO₄in acetonitrile/water=1:4/v:v pH 6.8; buffer B: 10 mM NaH₂PO₄, 1.5 MNaCl in acetonitrile/water=1:4/v:v; 5 to 60% B in 30 minutes at 1ml/min) or by capillary gel electrophoresis. The ODN can be purified byHPLC or by FPLC on a Source High Performance column (AmershamPharmacia). HPLC-homogeneous fractions are combined and desalted via aC18 column or by ultrafiltration. The ODN was analyzed by MALDI-TOF massspectrometry to confirm the calculated mass.

The nucleic acids of the invention can also include other modifications.These include nonionic DNA analogs, such as alkyl- and aryl-phosphates(in which the charged phosphonate oxygen is replaced by an alkyl or arylgroup), phosphodiester and alkylphosphotriesters, in which the chargedoxygen moiety is alkylated. Nucleic acids which contain diol, such astetraethyleneglycol or hexaethyleneglycol, at either or both terminihave also been shown to be substantially resistant to nucleasedegradation.

In some embodiments the oligonucleotides may be soft or semi-softoligonucleotides. A soft oligonucleotide is an immunostimulatoryoligonucleotide having a partially stabilized backbone, in whichphosphodiester or phosphodiester-like internucleotide linkages occuronly within and immediately adjacent to at least one internalpyrimidine-purine dinucleotide (YZ). Preferably YZ is YG, apyrimidine-guanosine (YG) dinucleotide. The at least one internal YZdinucleotide itself has a phosphodiester or phosphodiester-likeinternucleotide linkage. A phosphodiester or phosphodiester-likeinternucleotide linkage occurring immediately adjacent to the at leastone internal YZ dinucleotide can be 5′, 3′, or both 5′ and 3′ to the atleast one internal YZ dinucleotide.

In particular, phosphodiester or phosphodiester-like internucleotidelinkages involve “internal dinucleotides”. An internal dinucleotide ingeneral shall mean any pair of adjacent nucleotides connected by aninternucleotide linkage, in which neither nucleotide in the pair ofnucleotides is a terminal nucleotide, i.e., neither nucleotide in thepair of nucleotides is a nucleotide defining the 5′ or 3′ end of theoligonucleotide. Thus a linear oligonucleotide that is n nucleotideslong has a total of n-1 dinucleotides and only n-3 internaldinucleotides. Each internucleotide linkage in an internal dinucleotideis an internal internucleotide linkage. Thus a linear oligonucleotidethat is n nucleotides long has a total of n-1 internucleotide linkagesand only n-3 internal internucleotide linkages. The strategically placedphosphodiester or phosphodiester-like internucleotide linkages,therefore, refer to phosphodiester or phosphodiester-likeinternucleotide linkages positioned between any pair of nucleotides inthe nucleic acid sequence. In some embodiments the phosphodiester orphosphodiester-like internucleotide linkages are not positioned betweeneither pair of nucleotides closest to the 5′ or 3′ end.

Preferably a phosphodiester or phosphodiester-like internucleotidelinkage occurring immediately adjacent to the at least one internal YZdinucleotide is itself an internal internucleotide linkage. Thus for asequence N₁YZ N₂, wherein N₁ and N₂ are each, independent of the other,any single nucleotide, the YZ dinucleotide has a phosphodiester orphosphodiester-like internucleotide linkage, and in addition (a) N₁ andY are linked by a phosphodiester or phosphodiester-like internucleotidelinkage when N₁ is an internal nucleotide, (b) Z and N₂ are linked by aphosphodiester or phosphodiester-like internucleotide linkage when N₂ isan internal nucleotide, or (c) N₁ and Y are linked by a phosphodiesteror phosphodiester-like internucleotide linkage when N₁ is an internalnucleotide and Z and N₂ are linked by a phosphodiester orphosphodiester-like internucleotide linkage when N₂ is an internalnucleotide.

In the oligonucleotide of the invention at least one YZ of R₁YZR₂ mayhave a phosphodiester linkage. Alternatively the YZ of R₁YZR₂ may have aphosphorothioate linkage. In some embodiments the R₁Y and or ZR₂ ofR₁YZR₂ have a phosphorothioate linkage.

Soft oligonucleotides according to the instant invention are believed tobe relatively susceptible to nuclease cleavage compared to completelystabilized oligonucleotides. Without meaning to be bound to a particulartheory or mechanism, it is believed that soft oligonucleotides of theinvention are cleavable to fragments with reduced or noimmunostimulatory activity relative to fall-length soft oligonucleotide.Incorporation of at least one nuclease-sensitive internucleotidelinkage, particularly near the middle of the oligonucleotide, isbelieved to provide an “off switch” which alters the pharmacokinetics ofthe oligonucleotide so as to reduce the duration of maximalimmunostimulatory activity of the oligonucleotide. This can be ofparticular value in tissues and in clinical applications in which it isdesirable to avoid injury related to chronic local inflammation orimmunostimulation, e.g., the kidney.

A semi-soft oligonucleotide is an immunostimulatory oligonucleotidehaving a partially stabilized backbone, in which phosphodiester orphosphodiester-like internucleotide linkages occur only within at leastone internal pyrimidine-purine (YZ) dinucleotide. Semi-softoligonucleotides generally possess increased immunostimulatory potencyrelative to corresponding fully stabilized immunostimulatoryoligonucleotides. Due to the greater potency of semi-softoligonucleotides, semi-soft oligonucleotides may be used, in someinstances, at lower effective concentations and have lower effectivedoses than conventional fully stabilized immunostimulatoryoligonucleotides in order to achieve a desired biological effect.

It is believed that the foregoing properties of semi-softoligonucleotides generally increase with increasing “dose” ofphosphodiester or phosphodiester-like internucleotide linkages involvinginternal YZ dinucleotides. Thus it is believed, for example, thatgenerally for a given oligonucleotide sequence with five internal YZdinucleotides, an oligonucleotide with five internal phosphodiester orphosphodiester-like YZ internucleotide linkages is moreimmunostimulatory than an oligonucleotide with four internalphosphodiester or phosphodiester-like YG internucleotide linkages, whichin turn is more immunostimulatory than an oligonucleotide with threeinternal phosphodiester or phosphodiester-like YZ internucleotidelinkages, which in turn is more immunostimulatory than anoligonucleotide with two internal phosphodiester or phosphodiester-likeYZ internucleotide linkages, which in turn is more immunostimulatorythan an oligonucleotide with one internal phosphodiester orphosphodiester-like YZ internucleotide linkage. Importantly, inclusionof even one internal phosphodiester or phosphodiester-like YZinternucleotide linkage is believed to be advantageous over no internalphosphodiester or phosphodiester-like YZ internucleotide linkage. Inaddition to the number of phosphodiester or phosphodiester-likeinternucleotide linkages, the position along the length of the nucleicacid can also affect potency.

The soft and semi-soft oligonucleotides will generally include, inaddition to the phosphodiester or phosphodiester-like internucleotidelinkages at preferred internal positions, 5′ and 3′ ends that areresistant to degradation. Such degradation-resistant ends can involveany suitable modification that results in an increased resistanceagainst exonuclease digestion over corresponding unmodified ends. Forinstance, the 5′ and 3′ ends can be stabilized by the inclusion there ofat least one phosphate modification of the backbone. In a preferredembodiment, the at least one phosphate modification of the backbone ateach end is independently a phosphorothioate, phosphorodithioate,methylphosphonate, or methylphosphorothioate internucleotide linkage. Inanother embodiment, the degradation-resistant end includes one or morenucleotide units connected by peptide or amide linkages at the 3′ end.

A phosphodiester internucleotide linkage is the type of linkagecharacteristic of nucleic acids found in nature. The phosphodiesterinternucleotide linkage includes a phosphorus atom flanked by twobridging oxygen atoms and bound also by two additional oxygen atoms, onecharged and the other uncharged. Phosphodiester internucleotide linkageis particularly preferred when it is important to reduce the tissuehalf-life of the oligonucleotide.

A phosphodiester-like internucleotide linkage is a phosphorus-containingbridging group that is chemically and/or diastereomerically similar tophosphodiester. Measures of similarity to phosphodiester includesusceptibility to nuclease digestion and ability to activate RNAse H.Thus for example phosphodiester, but not phosphorothioate,oligonucleotides are susceptible to nuclease digestion, while bothphosphodiester and phosphorothioate oligonucleotides activate RNAse H.In a preferred embodiment the phosphodiester-like internucleotidelinkage is boranophosphate (or equivalently, boranophosphonate) linkage.U.S. Pat. Nos. 5,177,198; 5,859,231; 6,160,109; 6,207,819; Sagueev etal., (1998) J Am Chem Soc 120:9417-27. In another preferred embodimentthe phosphodiester-like internucleotide linkage is diasteromericallypure Rp phosphorothioate. It is believed that diasteromerically pure Rpphosphorothioate is more susceptible to nuclease digestion and is betterat activating RNAse H than mixed or diastereomerically pure Spphosphorothioate. Stereoisomers of CpG oligonucleotides are the subjectof co-pending U.S. patent application Ser. No. 09/361,575 filed Jul. 27,1999, and published PCT application PCT/US99/17100 (WO 00/06588). It isto be noted that for purposes of the instant invention, the term“phosphodiester-like internucleotide linkage” specifically excludesphosphorodithioate and methylphosphonate internucleotide linkages.

As described above the soft and semi-soft oligonucleotides of theinvention may have phosphodiester like linkages between C and G. Oneexample of a phosphodiester-like linkage is a phosphorothioate linkagein an Rp conformation. Oligonucleotide p-chirality can have apparentlyopposite effects on the immune activity of a CpG oligonucleotide,depending upon the time point at which activity is measured. At an earlytime point of 40 minutes, the R_(p) but not the S_(P) stereoisomer ofphosphorothioate CpG oligonucleotide induces JNK phosphorylation inmouse spleen cells. In contrast, when assayed at a late time point of 44hr, the S_(P) but not the R_(p) stereoisomer is active in stimulatingspleen cell proliferation. This difference in the kinetics andbioactivity of the R_(p) and S_(P) stereoisomers does not result fromany difference in cell uptake, but rather most likely is due to twoopposing biologic roles of the p-chirality. First, the enhanced activityof the Rp stereoisomer compared to the Sp for stimulating immune cellsat early time points indicates that the Rp may be more effective atinteracting with the CpG receptor, TLR9, or inducing the downstreamsignaling pathways. On the other hand, the faster degradation of the RpPS-oligonucleotides compared to the Sp results in a much shorterduration of signaling, so that the Sp PS-oligonucleotides appear to bemore biologically active when tested at later time points.

A surprisingly strong effect is achieved by the p-chirality at the CpGdinucleotide itself. In comparison to a stereo-random CpGoligonucleotide the congener in which the single CpG dinucleotide waslinked in Rp was slightly more active, while the congener containing anSp linkage was nearly inactive for inducing spleen cell proliferation.

The terms “nucleic acid” and “oligonucleotide” also encompass nucleicacids or oligonucleotides with substitutions or modifications, such asin the bases and/or sugars. For example, they include nucleic acidshaving backbone sugars that are covalently attached to low molecularweight organic groups other than a hydroxyl group at the 2′ position andother than a phosphate group or hydroxy group at the 5′ position. Thusmodified nucleic acids may include a 2′-O-alkylated ribose group. Inaddition, modified nucleic acids may include sugars such as arabinose or2′-fluoroarabinose instead of ribose. Thus the nucleic acids may beheterogeneous in backbone composition thereby containing any possiblecombination of polymer units linked together such as peptide-nucleicacids (which have an amino acid backbone with nucleic acid bases).

Nucleic acids also include substituted purines and pyrimidines such asC-5 propyne pyrimidine and 7-deaza-7-substituted purine modified bases.Wagner R W et al. (1996) Nat Biotechnol 14:840-4. Purines andpyrimidines include but are not limited to adenine, cytosine, guanine,thymine, 5-methylcytosine, 5-hydroxycytosine, 5-fluorocytosine,2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine,and other naturally and non-naturally occurring nucleobases, substitutedand unsubstituted aromatic moieties. Other such modifications are wellknown to those of skill in the art.

The immunostimulatory oligonucleotides of the instant invention canencompass various chemical modifications and substitutions, incomparison to natural RNA and DNA, involving a phosphodiesterinternucleotide bridge, a β-D-ribose unit and/or a natural nucleotidebase (adenine, guanine, cytosine, thymine, uracil). Examples of chemicalmodifications are known to the skilled person and are described, forexample, in Uhlmann B et al. (1990) Chem Rev 90:543; “Protocols forOligonucleotides and Analogs” Synthesis and Properties & Synthesis andAnalytical Techniques, S. Agrawal, Ed, Humana Press, Totowa, USA 1993;Crooke S T et al. (1996) Annu Rev Pharmacol Toxicol 36:107-129; andHunziker J et al. (1995) Mod Synth Methods 7:331-417. An oligonucleotideaccording to the invention may have one or more modifications, whereineach modification is located at a particular phosphodiesterinternucleotide bridge and/or at a particular β-D-ribose unit and/or ata particular natural nucleotide base position in comparison to anoligonucleotide of the same sequence which is composed of natural DNA orRNA.

For example, the invention relates to an oligonucleotide which maycomprise one or more modifications and wherein each modification isindependently selected from:

-   a) the replacement of a phosphodiester internucleotide bridge    located at the 3′ and/or the 5′ end of a nucleotide by a modified    internucleotide bridge,-   b) the replacement of phosphodiester bridge located at the 3′ and/or    the 5′ end of a nucleotide by a dephospho bridge,-   c) the replacement of a sugar phosphate unit from the sugar    phosphate backbone by another unit,-   d) the replacement of a β-D-ribose unit by a modified sugar unit,    and-   e) the replacement of a natural nucleotide base by a modified    nucleotide base.

More detailed examples for the chemical modification of anolignucleotide are as follows.

A phosphodiester internucleotide bridge located at the 3′ and/or the 5′end of a nucleotide can be replaced by a modified internucleotidebridge, wherein the modified internucleotide bridge is for exampleselected from phosphorothioate, phosphorodithioate,NR¹R²-phosphoramidate, boranophosphate, α-hydroxybenzyl phosphonate,phosphate-(C₁-C₂₁)—O-alkyl ester,phosphate-[(C₆-C₁₂)aryl-(C₁-C₂₁)—O-alkyl]ester, (C₁-C₈)alkylphosphonateand/or (C₆-C₁₂)arylphosphonate bridges, (C₇-C₁₂)-α-hydroxymethyl-aryl(e.g., disclosed in WO 95/01363), wherein (C₆-C₁₂)aryl, (C₆-C₂₀)aryl and(C₆-C₁₄)aryl are optionally substituted by halogen, alkyl, alkoxy,nitro, cyano, and where R¹ and R² are, independently of each other,hydrogen, (C₁-C₁₈)-alkyl, (C₆-C₂₀)-aryl, (C₆-C₁₄)-aryl-(C₁-C₈)-alkyl,preferably hydrogen, (C₁-C₈)-alkyl, preferably (C₁-C₄)-alkyl and/ormethoxyethyl, or R¹ and R² form, together with the nitrogen atomcarrying then, a 5-6-membered heterocyclic ring which can additionallycontain a further heteroatom from the group O, S and N.

The replacement of a phosphodiester bridge located at the 3′ and/or the5′ end of a nucleotide by a dephospho bridge (dephospho bridges aredescribed, for example, in Uhlmanm E and Peyman A in “Methods inMolecular Biology”, Vol. 20, “Protocols for Oligonucleotides andAnalogs”, S. Agrawal, Ed., Humana Press, Totowa 1993, Chapter 16, pp.355 ft), wherein a dephospho bridge is for example selected from thedephospho bridges formacetal, 3′-thioformacetal, methylhydroxylamine,oxime, methylenedimethyl-hydrazo, dimethylenesulfone and/or silylgroups.

A sugar phosphate unit (i.e., a β-D-ribose and phosphodiesterinternucleotide bridge together forming a sugar phosphate unit) from thesugar phosphate backbone (i.e., a sugar phosphate backbone is composedof sugar phosphate units) can be replaced by another unit, wherein theother unit is for example suitable to build up a “morpholino-derivative”oligomer (as described, for example, in Stirchak E P et al. (1989)Nucleic Acids Res 17:6129-41), that is, e.g., the replacement by amorpholino-derivative unit; or to build up a polyamide nucleic acid(“PNA”; as described for example, in Nielsen P E et al. (1994) BioconjugChem 5:3-7), that is, e.g., the replacement by a PNA backbone unit,e.g., by 2-aminoethylglycine.

A β-ribose unit or a β-D-2′-deoxyribose unit can be replaced by amodified sugar unit, wherein the modified sugar unit is for exampleselected from β-D-ribose, α-D-2′-deoxyribose, L-2′-deoxyribose,2′-F-2′-deoxyribose, 2′-F-arabinose, 2′-O—(C₁-C₆)alkyl-ribose,preferably 2′-O—(C₁-C₆)alkyl-ribose is 2′-O-methylribose,2′-O—(C₂-C₆)alkenyl-ribose, 2′-[O—(C₁-C₆)alkyl-O—(C₁-C₆)alkyl]-ribose,2′-NH₂-2′-deoxyribose, β-D-xylo-furanose, α-arabinofuranose,2,4-dideoxy-β-D-erythro-hexo-pyranose, and carbocyclic (described, forexample, in Froehler J (1992) Am Chem Soc 114:8320) and/or open-chainsugar analogs (described, for example, in Vandendriessche et al. (1993)Tetrahedron 49:7223) and/or bicyclosugar analogs (described, forexample, in Tarkov M et al. (1993) Helv Chim Acta 76:481).

In some preferred embodiments the sugar is 2′-O-methylribose,particularly for one or both nucleotides linked by a phosphodiester orphosphodiester-like internucleotide linkage.

Nucleic acids also include substituted purines and pyrimidines such asC-5 propyne pyrimidine and 7-deaza-7-substituted purine modified bases.Wagner R W et al. (1996) Nat Biotechnol 14:840-4. Purines andpyrimidines include but are not limited to adenine, cytosine, guanine,and thymine, and other naturally and non-naturally occurringnucleobases, substituted and unsubstituted aromatic moieties.

A modified base is any base which is chemically distinct from thenaturally occurring bases typically found in DNA and RNA such as T, C,G, A, and U, but which share basic chemical structures with thesenaturally occurring bases. The modified nucleotide base may be, forexample, selected from hypoxanthine, uracil, dihydrouracil,pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil,5-(C₁-C₆)-alkyluracil, 5-(C₂-C₆)-alkenyluracil, 5-(C₂-C₆)-alkynyluracil,5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil,5-hydroxycytosine, 5-(C₁-C₆)-alkylcytosine, 5-(C₂-C₆)-alkenylcytosine,5-(C₂-C₆)-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine,5-bromocytosine, N²-dimethylguanine, 2,4-diamino-purine, 8-azapurine, asubstituted 7-deazapurine, preferably 7-deaza-7-substituted and/or7-deaza-8-substituted purine, 5-hydroxymethylcytosine, N4-alkylcytosine,e.g., N4-ethylcytosine, 5-hydroxydeoxycytidine,5-hydroxymethyldeoxycytidine, N4-alkyldeoxycytidine, e.g.,N4-ethyldeoxycytidine, 6-thiodeoxyguanosine, and deoxyribonucleotides ofnitropyrrole, C5-propynylpyrimidine, and diaminopurine e.g.,2,6-diaminopurine, inosine, 5-methylcytosine, 2-aminopurine,2-amino-6-chloropurine, hypoxanthine or other modifications of a naturalnucleotide bases. This list is meant to be exemplary and is not to beinterpreted to be limiting.

In particular formulas described herein a set of modified bases isdefined. For instance the letter Y is used to refer to pyrimidine and insome embodiments a nucleotide containing a cytosine or a modifiedcytosine. A modified cytosine as used herein is a naturally occurring ornon-naturally occurring pyrimidine base analog of cytosine which canreplace this base without impairing the immunostimulatory activity ofthe oligonucleotide. Modified cytosines include but are not limited to5-substituted cytosines (e.g. 5-methyl-cytosine, 5-fluoro-cytosine,5-chloro-cytosine, 5-bromocytosine 5-iodo-cytosine, 5-hydroxy-cytosine,5-hydroxymethyl-cytosine, 5-difluoromethyl-cytosine, and unsubstitutedor substituted 5-alkynyl-cytosine), 6-substituted cytosines,N4-substituted cytosines (e.g. N4-ethyl-cytosine), 5-aza-cytosine,2-mercapto-cytosine, isocytosine, pseudo-isocytosine, cytosine analogswith condensed ring systems (e.g. N,N′-propylene cytosine orphenoxazine), and uracil and its derivatives (e.g. 5-fluoro-uracil,5-bromo-uracil, 5-bromovinyl-uracil, 4-thio-uracil, 5-hydroxy-uracil,5-propynyl-uracil). Some of the preferred cytosines include5-methyl-cytosine, 5-fluoro-cytosine, 5-hydroxy-cytosine,5-hydroxymethyl-cytosine, and N4-ethyl-cytosine. In another embodimentof the invention, the cytosine base is substituted by a universal base(e.g. 3-nitropyrrole, P-base), an aromatic ring system (e.g.fluorobenzene or difluorobenzene) or a hydrogen atom (dSpacer).

The letter Z is used to refer to a purine, pyrimidine, or abasic and insome embodiments a guanine or a modified guanine base. A modifiedguanine as used herein is a naturally occurring or non-naturallyoccurring purine base analog of guanine which can replace this basewithout impairing the immunostimulatory activity of the oligonucleotide.Modified guanines include but are not limited to 7-deazaguanine,7-deaza-7-substituted guanine (such as 7-deaza-7-(C2-C6)alkynylguanine),7-deaza-8-substituted guanine, hypoxanthine, N2-substituted guanines(e.g. N2-methyl-guanine),5-amino-3-methyl-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione,2,6-diaminopurine, 2-aminopurine, purine, indole, adenine, substitutedadenines (e.g. N6-methyl-adenine, 8-oxo-adenine) 8-substituted guanine(e.g. 8-hydroxyguanine and 8-bromoguanine) and 6-thioguanine. In anotherembodiment of the invention, the guanine base is substituted by auniversal base (e.g. 4-methyl-indole, 5-nitro-indole, and K-base), anaromatic ring system (e.g. benzimidazole or dichloro-benzimidazole,1-methyl-1H-[1,2,4]triazolo-3-carboxylic acid amide) or a hydrogen atom(dSpacer).

The oligonucleotides may have one or more accessible 5′ ends. It ispossible to create modified oligonucleotides having two such 5′ ends.This may be achieved, for instance by attaching two oligonucleotidesthrough a 3′-3′ linkage to generate an oligonucleotide having one or twoaccessible 5′ ends. The 33′-linkage may be a phosphodiester,phosphorothioate or any other modified internucleotide bridge. Methodsfor accomplishing such linkages are known in the art. For instance, suchlinkages have been described in Seliger, H.; et al., Oligonucleotideanalogs with terminal 3′-3′- and 5′-5′-internucleotidic linkages aantisense inhibitors of viral gene expression, Nucleotides & Nucleotides(1991), 10(1-3), 469-77 and Jiang, et al., Pseudo-cyclicoligonucleotides: in vitro and in vivo properties, Bioorganic &Medicinal Chemistry (1999), 7(12), 2727-2735.

Additionally, 3′3′-linked nucleic acids where the linkage between the3′-terminal nucleotides is not a phosphodiester, phosphorothioate orother modified bridge, can be prepared using an additional spacer, suchas tri- or tetra-ethylenglycol phosphate moiety (Durand, M. et al,Triple-helix formation by an oligonucleotide containing one (dA)12 andtwo (dT)12 sequences bridged by two hexaethylene glycol chains,Biochemistry (1992), 31(38), 9197-204, U.S. Pat. No. 5,658,738, and U.S.Pat. No. 5,668,265). Alternatively, the non-nucleotidic linker may bederived from ethanediol, propanediol, or from an abasic deoxyribose(dSpacer) unit (Fontanel, Marie Laurence et al., Sterical recognition byT4 polynucleotide kinase of non-nucleosidic moieties 5′-attached tooligonucleotides; Nucleic Acids Research (1994), 22(11), 2022-7) usingstandard phosphoramidite chemistry. The non-nucleotidic linkers can beincorporated once or multiple times, or combined with each otherallowing for any desirable distance between the 3′-ends of the two ODNsto be linked.

The oligonucleotides are partially resistant to degradation (e.g., arestabilized). A “stabilized oligonucleotide molecule” shall mean anoligonucleotide that is relatively resistant to in vive degradation(e.g. via an exo- or endo-nuclease). Nucleic acid stabilization can beaccomplished via backbone modifications. Oligonucleotides havingphosphorothioate linkages provide maximal activity and protect theoligonucleotide from degradation by intracellular exo- andendo-nucleases. Other modified oligonucleotides include phosphodiestermodified nucleic acids, combinations of phosphodiester andphosphorothioate nucleic acid, methylphosphonate,methylphosphorothioate, phosphorodithioate, p-ethoxy, and combinationsthereof:

Modified backbones such as phosphorothioates may be synthesized usingautomated techniques employing either phosphoramidate or H-phosphonatechemistries. Aryl- and alkyl-phosphonates can be made, e.g., asdescribed in U.S. Pat. No. 4,469,863; and alkylphosphotriesters (inwhich the charged oxygen moiety is alkylated as described in U.S. Pat.No. 5,023,243 and European Patent No. 092,574) can be prepared byautomated solid phase synthesis using commercially available reagents.Methods for making other DNA backbone modifications and substitutionshave been described (e.g., Uhlmann, B. and Peyman, A., Chem. Rev.90:544, 1990; Goodchild, J., Bioconjugate Chem. 1:165, 1990).

Other stabilized oligonucleotides include: nonionic DNA analogs, such asalkyl- and aryl-phosphates (in which the charged phosphonate oxygen isreplaced by an alkyl or aryl group), phosphodiester andalkylphosphotriesters, in which the charged oxygen moiety is alkylated.Nucleic acids which contain diol, such as tetraethyleneglycol orhexaethyleneglycol, at either or both termini have also been shown to besubstantially resistant to nuclease degradation.

In some embodiments the oligonucleotide comprises one or morepalindromic sequences. As used herein, “palindrome” and, equivalently,“palindromic sequence” shall refer to an inverted repeat, i.e., asequence such as ABCDEE′D′C′B′A′ in which A and A′, B and B′, etc., arebases capable of forming the usual Watson-Crick base pairs. In somecases the palindrome is GC-rich. A GC-rich palindrome is a palindromehaving a base composition of at least two-thirds G's and C's. In someembodiments the GC-rich domain is preferably 3′ to the “B cellstimulatory domain”. In the case of a 10-base long GC-rich palindrome,the palindrome thus contains at least 8 G's and C's. In the case of a12-base long GC-rich palindrome, the palindrome also contains at least 8G's and C's. In the case of a 14-mar GC-rich palindrome, at least tenbases of the palindrome are G's and C's. In some embodiments the GC-richpalindrome is made up exclusively of G's and C's. In some embodimentsthe oligonucleotide contains more than one palindromic sequence.

DNA is a polymer of deoxyribonucleotides joined through 3′-5′phosphodiester linkages. Units of the polymer of the invention can alsobe joined through 3′-5′ phosphodiester linkages. However, the inventionalso encompasses polymers having unusual internucleotide linkages,including specifically 5′-5′, 3′-3′, 2′-2′, 2′-3′, and 2′-5′internucleotide linkages. In one embodiment such unusual linkages areexcluded from the immunostimulatory DNA motif, even though one or moreof such linkages may occur elsewhere within the polymer. For polymershaving free ends, inclusion of one 3′-3′ internucleotide linkage canresult in a polymer having two free 5′ ends. Conversely, for polymershaving free ends, inclusion of one 5′-5′ internucleotide linkage canresult in a polymer having two free 3′ ends.

An immunostimulatory composition of this invention can contain two ormore immunostimulatory DNA motifs which can be linked through abranching unit. The internucleotide linkages can be 3′-5′, 5′-5′, 3′-3′,2′-2′, 2′-3′, or 2′-5′ linkages. Thereby, the nomenclature 2′-5′ ischosen according to the carbon atom of deoxyribose. However, ifunnatural sugar moieties are employed, such as ring-expanded sugaranalogs (e.g., hexanose, cylohexene or pyranose) or bi- or tricyclicsugar analogs, then this nomenclature changes according to thenomenclature of the monomer. The unusual internucleotide linkage can bea phosphodiester linkage, but it can alternatively be modified asphosphorothioate or any other modified linkage as described herein.Formula IV shows a general structure for branched DNA oligomers andmodified oligoribonucleotide analogs of the invention via a nucleotidicbranching unit. Thereby Nu₁, Nu₂, and Nu₃ can be linked through 3′-5′,5′-5′, 3′-3′, 2-2′, 2′-3′, or 2′-5′-linkages. Branching of DNA oligomerscan also involve the use of non-nucleotidic linkers and abasic spacers.In one embodiment, Nu₁, Nu₂, and Nu₃ represent identical or differentimmunostimulatory DNA motifs.

The modified oligoribonucleotide analog may contain a doubler or treblerunit (Glen Research. Sterling, Va.), in particular those modifiedoligodeoxyribonucleotide analogs with a 3′-3′ linkage. A doubler unit inone embodiment can be based on1,3-bis-[5-(4,4′-dimethoxytrityloxy)pentylamido]propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.A trebler unit in one embodiment can be based on incorporation ofTris-2,2,2-[3-(4,4′-dimethoxytrityloxy)propyloxymethyl]ethyl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.Branching of the modified oligoribonucleotide analogs by multipledoubler, trebler, or other multiplier units leads to dendrimers whichare a farther embodiment of this invention. Branched modifiedoligoribonucleotide analogs may lead to crosslinking of receptorsparticularly for combinations of immunostimulatory RNA and DNA such asTLR3, TLR7, TLR8, and TLR9 with distinct immune effects compared tonon-branched forms of the analogs. In addition, the synthesis ofbranched or otherwise multimeric analogs may stabilize DNA againstdegradation and may enable weak or partially effective DNA sequences toexert a therapeutically useful level of immune activity. The modifiedoligodeoxyribonucleotide analogs may also contain linker units resultingfrom peptide modifying reagents or oligonucleotide modifying reagents(Glen Research). Furthermore, the modified oligodeoxyribonucleotideanalogs may contain one or more natural or unnatural amino acid residueswhich are connected to the polymer by peptide (amide) linkages.

The 3′-5′, 5′-5′, 3′-3′, 2′-2′, 2′-3′, and 2′-5′ internucleotidelinkages can be direct or indirect. Direct linkages in this contextrefers to a phosphate or modified phosphate linkage as disclosed herein,without an intervening linker moiety. An intervening linker moiety is anorganic moiety distinct from a phosphate or modified phosphate linkageas disclosed herein, which can include, for example, polyethyleneglycol, triethylene glycol, hexaethylene glycol, dSpacer (i.e., anabasic deoxynucleotide), doubler unit, or trebler unit.

The linkages are preferably composed of C, H, N, O, S, B, P, andHalogen, containing 3 to 300 atoms. An example with 3 atoms is an acetallinkage (ODN1-3′-O—CH₂—O-3′-ODN2) connecting e.g. the 3′-hydroxy groupof one nucleotide to the 3′-hydroxy group of a second oligonucleotide.An example with about 300 atoms is PEG-40 (tetracontapolyethyleneglycol). Preferred linkages are phosphodiester,phosphorothioate, methylphosphonate, phosphoramidate, boranophosphonate,amide, ether, thioether, acetal, thioacetal, urea, thiourea,sulfonamide, Schiff Base and disulfide linkages. It is also possible touse the Solulink BioConjugation System i.e., (www.trilinkbiotech.com).

If the oligonucleotide is composed of two or more sequence parts, theseparts can be identical or different. Thus, in an oligonucleotide with a3′3′-linkage, the sequences can be identical 5′-ODN1-3′3′-ODN1-5′ ordifferent 5′-ODN1-3′3′-ODN2-5′. Furthermore, the chemical modificationof the various oligonucleotide parts as well as the linker connectingthem may be different. Since the uptake of short oligonucleotidesappears to be less efficient than that of long oligonucleotides, linkingof two or more short sequences results in improved immune stimulation.The length of the short oligonucleotides is preferably 2-20 nucleotides,more preferably 3-16 nucleotides, but most preferably 5-10 nucleotides.Preferred are linked oligonucleotides which have two or more unlinked5′-ends.

The oligonucleotide partial sequences may also be linked bynon-nucleotidic linkers. A “non-nucleotidic linker” as used hereinrefers to any linker element that is not a nucleotide or polymer thereof(i.e., a polynucleotide), wherein a nucleotide includes a purine orpyrimidine nucleobase and a sugar phosphate, in particular abasiclinkers (dSpacers), trietyhlene glycol units or hexaethylene glycolunits. Further preferred linkers are alkylamino linkers, such as C3, C6,C12 aminolinkers, and also alkylthiol linkers, such as C3 or C6 thiollinkers. The oligonucleotides can also be linked by aromatic residueswhich may be further substituted by alkyl or substituted alkyl groups.

For facilitating uptake into cells, the immunostimulatoryoligonucleotides are in some embodiments in the range of 3 to 100 basesin length. In some embodiments the oligonucleotides are 7-100 bases inlength. Typically, nucleic acids of any size greater than 6 nucleotides(even many kb long) are capable of inducing an immune response accordingto the invention if sufficient immunostimulatory motifs are present.However, the improved immunostimulatory capacity of the modifiedoligonucleotides of the invention provides for immunostimulatorymolecules of much shorter length. In some embodiments theimmunostimulatory oligonucleotides are 3-6 bases in length.

The CpG immunostimulatory oligonucleotides are useful in some aspects ofthe invention as a vaccine for the treatment of a subject at risk ofdeveloping allergy or asthma, an infection with an infectious organismor a cancer in which a specific cancer antigen has been identified. TheCpG immunostimulatory oligonucleotides can also be given without theantigen or allergen for protection against infection, allergy or cancer,and in this case repeated doses may allow longer team protection. Asubject at risk as used herein is a subject who has any risk of exposureto an infection causing pathogen or a cancer or an allergen or a risk ofdeveloping cancer. For instance, a subject at risk may be a subject whois planning to travel to w area where a particular type of infectiousagent is found or it may be a subject who through lifestyle or medicalprocedures is exposed to bodily fluids which may contain infectiousorganisms or directly to the organism or even any subject living in anarea where an infectious organism or an allergen has been identified.Subjects at risk of developing infection also include generalpopulations to which a medical agency recommends vaccination with aparticular infectious organism antigen. If the antigen is an allergenand the subject develops allergic responses to that particular antigenand the subject may be exposed to the antigen, i.e., during pollenseason, then that subject is at risk of exposure to the antigen. Asubject at risk of developing allergy or asthma includes those subjectsthat have been identified as having an allergy or asthma but that don'thave the active disease during the CpG immunostimulatory oligonucleotidetreatment as well as subjects that are considered to be at risk ofdeveloping these diseases because of genetic or environmental factors.

A subject at risk of developing a cancer is one who has a highprobability of developing cancer. These subjects include, for instance,subjects having a genetic abnormality, the presence of which has beendemonstrated to have a correlative relation to a higher likelihood ofdeveloping a cancer and subjects exposed to cancer causing agents suchas tobacco, asbestos, or other chemical toxins, or a subject who haspreviously been treated for cancer and is in apparent remission. When asubject at risk of developing a cancer is treated with an antigenspecific for the type of cancer to which the subject is at risk ofdeveloping and a CpG immunostimulatory oligonucleotide, the subject maybe able to kill the cancer cells as they develop. If a tumor begins toform in the subject, the subject will develop a specific immune responseagainst the tumor antigen.

In addition to the use of the CpG immunostimulatory oligonucleotides forprophylactic treatment, the invention also encompasses the use of theCpG immunostimulatory oligonucleotides for the treatment of a subjecthaving an infection, an allergy, asthma, or a cancer.

A subject having an infection is a subject that has been exposed to aninfectious pathogen and has acute or chronic detectable levels of thepathogen in the body. The CpG immunostimulatory oligonucleotides can beused with or without an antigen to mount an antigen specific systemic ormucosal immune response that is capable of reducing the level of oreradicating the infectious pathogen. An infectious disease, as usedherein, is a disease arising from the presence of a foreignmicroorganism in the body. It is particularly important to developeffective vaccine strategies and treatments to protect the body'smucosal surfaces, which are the primary site of pathogenic entry.

A subject having an allergy is a subject that has or is at risk ofdeveloping an allergic reaction in response to an allergen. An allergyrefers to acquired hypersensitivity to a substance (allergen). Allergicconditions include but are not limited to eczema, allergic rhinitis orcoryza, bay fever, conjunctivitis, bronchial asthma, urticaria (hives)and food allergies, and other atopic conditions.

Allergies are generally caused by IgE antibody generation againstharmless allergens. The cytokines that are induced by systemic ormucosal administration of CpG immunostimulatory oligonucleotides arepredominantly of a class called Th1 (examples are IL-12, IP-10, IFN-αand IFN-γ) and these induce both humoral and cellular immune responses.The other major type of immune response, which is associated with theproduction of IL-4 and IL-5 cytokines, is termed a Th2 immune response.In general, it appears that allergic diseases are mediated by Th2 typeimmune responses. Based on the ability of the CpG immunostimulatoryoligonucleotides to shift the immune response in a subject from apredominant Th2 (which is associated with production of IgE antibodiesand allergy) to a balanced Th2/Th1 response (which is protective againstallergic reactions), an effective dose for inducing an immune responseof a CpG immunostimulatory oligonucleotide can be administered to asubject to treat or prevent asthma and allergy.

Thus, the CpG immunostimulatory oligonucleotides have significanttherapeutic utility in the treatment of allergic and non-allergicconditions such as asthma. Th2 cytokines, especially IL-4 and IL-5 areelevated in the airways of asthmatic subjects. These cytokines promoteimportant aspects of the asthmatic inflammatory response, including IgEisotope switching, eosinophil chemotaxis and activation and mast cellgrowth. Th1 cytokines, especially IFN-γ and IL-12, can suppress theformation of Th2 clones and production of Th2 cytokines. Asthma refersto a disorder of the respiratory system characterized by inflammation,narrowing of the airways and increased reactivity of the airways toinhaled agents. Asthma is frequently, although not exclusivelyassociated with atopic or allergic symptoms.

A subject having a cancer is a subject that has detectable cancerouscells. The cancer may be a malignant or non-malignant cancer. Cancers ortumors include but are not limited to biliary tract cancer, braincancer; breast cancer, cervical cancer, choriocarcinoma; colon cancer;endometrial cancer; esophageal cancer; gastric cancer; intraepithelialneoplasms; lymphomas; liver cancer, lung cancer (e.g. small cell andnon-small cell); melanoma; neuroblastomas; oral cancer, ovarian cancer;pancreas cancer; prostate cancer rectal cancer sarcomas; skin cancer;testicular cancer, thyroid cancer; and renal cancer, as well as othercarcinomas and sarcomas. In one embodiment the cancer is hairy cellleukemia, chronic myelogenous leukemia, cutaneous T-cell leukemia,multiple myeloma, follicular lymphoma, malignant melanoma, squamous cellcarcinoma, renal cell carcinoma, prostate carcinoma, bladder cellcarcinoma, or colon carcinoma.

A subject shall mean a human or vertebrate animal including but notlimited to a dog, cat, horse, cow, pig, sheep, goat, turkey, chicken,primate, e.g., monkey, and fish (aquaculture species), e.g. salmon.Thus, the invention can also be used to treat cancer and tumors,infections, and allergy/asthma in non human subjects. Cancer is one ofthe leading causes of death in companion animals (i.e., cats and dogs).

As used herein, the term treat, treated, or treating when used withrespect to an disorder such as an infectious disease, cancer, allergy,or asthma refers to a prophylactic treatment which increases theresistance of a subject to development of the disease (e.g., toinfection with a pathogen) or, in other words, decreases the likelihoodthat the subject will develop the disease (e.g., become infected withthe pathogen) as well as a treatment after the subject has developed thedisease in order to fight the disease (e.g., reduce or eliminate theinfection) or prevent the disease from becoming worse.

In the instances when the CpG oligonucleotide is administered with anantigen, the subject may be exposed to the antigen. As used herein, theterm exposed to refers to either the active step of contacting thesubject with an antigen or the passive exposure of the subject to theantigen in vivo. Methods for the active exposure of a subject to anantigen are well-known in the art. In general, an antigen isadministered directly to the subject by any means such as intravenous,intramuscular, oral, transdermal, mucosal, intranasal, intratracheal, orsubcutaneous administration. The antigen can be administeredsystemically or locally. Methods for administering the antigen and theCpG immunostimulatory oligonucleotide are described in more detailbelow. A subject is passively exposed to an antigen if an antigenbecomes available for exposure to the immune cells in the body. Asubject may be passively exposed to an antigen, for instance, by entryof a foreign pathogen into the body or by the development of a tumorcell expressing a foreign antigen on its surface.

The methods in which a subject is passively exposed to an antigen can beparticularly dependent on timing of administration of the CpGimmunostimulatory oligonucleotide. For instance, in a subject at risk ofdeveloping a cancer or an infectious disease or an allergic or asthmaticresponse, the subject may be administered the CpG immunostimulatoryoligonucleotide on a regular basis when that risk is greatest, i.e.,during allergy season or after exposure to a cancer causing agent.Additionally the CpG immunostimulatory oligonucleotide may beadministered to travelers before they travel to foreign lands where theyare at risk of exposure to infections agents. Likewise the CpGimmunostimulatory oligonucleotide may be administered to soldiers orcivilians at risk of exposure to biowarfare to induce a systemic ormucosal immune response to the antigen when and if the subject isexposed to it.

An antigen as used herein is a molecule capable of provoking an immuneresponse. Antigens include but are not limited to cells, cell extracts,proteins, polypeptides, peptides, polysaccharides, polysaccharideconjugates, peptide and non-peptide mimics of polysaccharides and othermolecules, small molecules, lipids, glycolipids, carbohydrates, virusesand viral extracts and multicellular organisms such a parasites andallergens. The term antigen broadly includes any type of molecule whichis recognized by a host immune system as being foreign. Antigens includebut are not limited to cancer antigens, microbial antigens, andallergens.

A cancer antigen as used herein is a compound, such as a peptide orprotein, associated with a tumor or cancer cell surface and which iscapable of provoking an immune response when expressed on the surface ofan antigen presenting cell in the context of an MHC molecule. Cancerantigens can be prepared from cancer cells either by preparing crudeextracts of cancer cells, for example, as described in Cohen, et al.,1994, Cancer Research, 54:1055, by partially purifying the antigens, byrecombinant technology, or by de novo synthesis of known antigens.Cancer antigens include but are not limited to antigens that arerecombinantly expressed, an immunogenic portion of, or a whole tumor orcancer. Such antigens can be isolated or prepared recombinantly or byany other means known in the art.

A microbial antigen as used herein is an antigen of a microorganism andincludes but is not limited to virus, bacteria, parasites, and fungi.Such antigens include the intact microorganism as well as naturalisolates and fragments or derivatives thereof and also syntheticcompounds which are identical to or similar to natural microorganismantigens and induce an immune response specific for that microorganism.A compound is similar to a natural microorganism antigen if it inducesan immune response (humoral and/or cellular) to a natural microorganismantigen. Such antigens are used routinely in the at and are well knownto those of ordinary skill in the art.

Viruses as small infectious agents which generally contain a nucleicacid core and a protein coat, but are not independently livingorganisms. Viruses can also take the form of infectious nucleic acidslacking a protein. A virus cannot survive in the absence of a livingcell within which it can replicate. Viruses enter specific living cellseither by endocytosis or direct injection of DNA (phage) and multiply,causing disease. The multiplied virus can then be released and infectadditional cells. Some viruses are DNA-containing viruses and others areRNA-containing viruses. DNA viruses include Pox, Herpes, Adeno, Papova,Parvo, and Hepadna. RNA viruses include Picorna, Calici, Astro, Toga,Flavi, Corona, Paramyxo, Orthomyxo, Bunya, Arena, Rhabdo, Filo, Borna,Reo, and Retro. In some aspects, the invention also intends to treatdiseases in which prions are implicated in disease progression such asfor example bovine spongiform encephalopathy (i.e., mad cow disease,BSE) or scrapie infection in animals, or Creutzfeldt-Jakob disease inhumans.

Viruses include, but are not limited to, enteroviruses (including, butnot limited to, viruses that the family picornaviridae, such as poliovirus, Coxsackie virus, echo virus), rotaviruses, adenovirus, andhepatitis virus, such as hepatitis A, B, C D and E. Specific examples ofviruses that have been found in humans include but are not limited to:Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (alsoreferred to as HTLV-III, LAV or HTLV-II/LAV, or HIV-III; and otherisolates, such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitisA virus; enteroviruses, human Coxsackie viruses, rhinoviruses,echoviruses); Caldviridae (e.g., strains that cause gastroenteritis);Togaviridae (e.g., equine encephalitis viruses, rubella viruses);Flaviviridae (e.g., dengue viruses, encephalitis viruses, yellow feverviruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g.,vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., ebolaviruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus,measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g.,influenza viruses); Bunyaviridae (e.g., Hantaan viruses, bunya viruses,phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic feverviruses); Reoviridae (e.g., reoviruses, orbiviruses and rotaviruses);Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae(parvoviruses); Papovaviridae (papillomaviruses, polyoma viruses);Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus(HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV));Poxviridae (variola viruses, vaccinia viruses, pox viruses);Iridoviridae (e.g., African swine fever virus); and other viruses acutelaryngotracheobronchitis virus, Alphavirus, Kaposi's sarcoma-associatedherpesvirus, Newcastle disease virus, Nipah virus, Norwalk virus,Papillomavirus, parainfluenza virus, avian influenza, SARs virus, WestNile virus.

The methods of the invention are particularly useful, in someembodiments, for the treatment of Human immunodeficiency virus (HIV) andhepatitis virus. HIV, a species of retrovirus also known as human T-celllymphotropic virus III (HTLV III), is responsible for causing thedeterioration resulting in the disorder known as AIDS. HIV infects anddestroys T-cells, upsetting the overall balance of the immune system,resulting in a loss in the patients ability to combat other infectionsand predisposing the patient to opportunistic infections whichfrequently prove fatal.

Viral hepatitis is an inflammation of the liver which may produceswelling, tenderness, and sometimes permanent damage to the liver. Ifthe inflammation of the liver continues at least six months or longer,it is referred to as chronic hepatitis. There are at least fivedifferent viruses known to cause viral hepatitis, include hepatitis A,B, C D and E. Hepatitis A is generally communicated through food ordrinking water contaminated with human feces. Hepatitis B generally isspread through bodily fluids such as blood. For instance, it may bespread from mother to child at birth, through sexual contact,contaminated blood transfusions and needles. Hepatitis C is quite commonand like Hepatitis B is often spread through blood transfusions andcontaminated needles. Hepatitis D is found most often in IV drug userswho are carriers of the hepatitis B virus with which it co-associates.Hepatitis B is similar to viral hepatitis A and is generally associatedwith poor sanitation.

Both gram negative and gram positive bacteria serve as antigens invertebrate animals. Such gram positive bacteria include, but are notlimited to, Pasteurella species, Staphylococci species, andStreptococcus species. Gram negative bacteria include, but are notlimited to, Escherichia coli, Pseudomonas species, and Salmonellaspecies. Specific examples of infectious bacteria include but are notlimited to, Helicobacter pyloris, Borelia burgdorferi, Legionellapneumophilia, Mycobacteria sps (e.g. M. tuberculosis, M. avium, M.intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus,Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes,Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae(Group B Streptococcus), Streptococcus (viridans group). Streptococcusfaecalis, Streptococcus bovis, Streptococcus (anaerobic sps.),Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococus sp.,Haemophilus influenza, Bacillus antracis, corynebacterium diphtheriae,corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridiumperfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiellapneumonirae, Pasturella multocida, Bacteroides sp., Fusobacteriumnucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponemapertenue, Leptospira, Rickettsia and Actinomyces israelli.

Examples of fungi include Cryptococcus neoformans, Histoplasmacapsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydiatrachomatis, Candida albicans.

Other infectious organisms (i.e., protists) include Plasmodium spp. suchas Plasmodium falciparum, Plasmodium malariae, Plosmodium ovale, andPlasmodium vivax and Toxoplasma gondii. Blood-borne and/or tissuesparasites include Plasmodium spp., Babesia microti, Babesia divergens,Leishmania tropica, Leishmania spp., Leishmania braziliensis, Leishmaniadonovani, Trypanosoma gambiense and Trypanosoma rhodesiense (Africansleeping sickness), Trypanosoma cruzi (Chagas' disease), and Toxoplasmagondii.

Other medically relevant microorganisms have been described extensivelyin the literature, e.g., see C. G. A Thomas, Medical Microbiology,Bailliere Tindall, Great Britain 1983, the entire contents of which ishereby incorporated by reference.

An allergen refers to a substance (antigen) that can induce an allergicor asthmatic response in a susceptible subject. The list of allergens isenormous and can include pollens, insect venoms, animal dander dust,fungal spores and drugs (e.g. penicillin). Examples of natural, animaland plant allergens include but are not limited to proteins specific tothe following genuses: Canine (Canis familiaris); Dermatophagoides (e.g.Dermatophagoides farinae); Felis (Felis domesticus); Ambrosia (Ambrosiaartemiisfolia; Lolium (e.g. Lolium perenne or Lolium multiflorum);Cryptomeria (Cryptomeria japonica); Alternaria (Alternaria alternata);Alder, Alnus (Alnus gultinoasa); Betula (Betula verrucosa); Quercus(Quercus alba); Olea (Olea europa); Artemisia (Artemisia vulgaris);Plantago (e.g. Plantago lanceolata); Parietaria (e.g. Parietariaofficinalis or Parietaria judaica); Blattella (e.g. Blattellagermanica); Apis (e.g. Apis multiflorum); Cupressus (e.g. Cupressussempervirens, Cupressus arizonica and Cupressus macrocarpa); Juniperus(e.g. Juniperus sabinoides, Juniperus virginiana, Juniperus communis andJuniperus ashei); Thuya (e.g. Thuya orientalis); Chamaecyparis (e.g.Chamaecyparis obtusa); Periplaneta (e.g. Periplaneta americana);Agropyron (e.g. Agropyron repens); Secale (e.g. Secale cereale); Tritcum(e.g. Tritcum aestivum); Dactylis (e.g. Dactylis glomerata); Festuca(e.g. Festuca elatior); Poa (e.g. Poa pratensis or Poa compressa); Avena(e.g. Avena sativa); Holcus (e.g. Holcus lanatus); Anthoxanthum (e.g.Anthoxanthum odoratum); Arrhenatherum (e.g. Arrhenatherum elatius);Agrostis (e.g. Agrostis alba); Phleum (e.g. Phleum pratense); Phalaris(e.g. Phalaris arundinacea); Paspalum (e.g. Paspalum notatum); Sorghum(e.g. Sorghum halepensis); and Bromus (e.g. Bromus inermis).

The term substantially purified as used herein refers to a polypeptidewhich is substantially free of other proteins, lipids, carbohydrates orother materials with which it is naturally associated. One skilled inthe art can purify viral or bacterial polypeptides using standardtechniques for protein purification. The substantially pure polypeptidewill often yield a single major band on a non-reducing polyacrylamidegel. In the case of partially glycosylated polypeptides or those thathave several start codons, there may be several bands on a non-reducingpolyacrylamide gel, but these will form a distinctive pattern for thatpolypeptide. The purity of the viral or bacterial polypeptide can alsobe determined by amino-terminal amino acid sequence analysis. Othertypes of antigens not encoded by a nucleic acid vector such aspolysaccharides, small molecule, mimics etc are included within theinvention.

The oligonuclotides of the invention may be administered to a subjectwith an anti-microbial agent. An anti-microbial agent, as used herein,refers to a naturally-occurring or synthetic compound which is capableof killing or inhibiting infectious microorganisms. The type ofanti-microbial agent useful according to the invention will depend uponthe type of microorganism with which the subject is infected or at riskof becoming infected. Anti-microbial agents include but are not limitedto anti-bacterial agents, anti-viral agents, anti-fungal agents andanti-parasitic agents. Phrases such as “anti-infective agent”,“anti-bacterial agent”, “anti-viral agent”, “anti-fungal agent”,“anti-parasitic agent” and “parasiticide” have well-established meaningsto those of ordinary skill in the art and are defined in standardmedical texts. Briefly, anti-bacterial agents kill or inhibit bacteria,and include antibiotics as well as other synthetic or natural compoundshaving similar functions. Antibiotics are low molecular weight moleculeswhich are produced as secondary metabolites by cells, such asmicroorganisms. In general, antibiotics interfere with one or morebacterial functions or structures which are specific for themicroorganism and which are not present in host cells. Anti-viral agentscan be isolated from natural sources or synthesized and are useful forkilling or inhibiting viruses. Anti-fungal agents are used to treatsuperficial fungal infections as well as opportunistic and primarysystemic fungal infections. Anti-parasite agents kill or inhibitparasites.

Examples of anti-parasitic agents, also referred to as parasiticidesuseful for human administration include but are not limited toalbendazole, amphotericin B, benzidazole, bithionol, chloroquine HCl,chloroquine phosphate, clindamycin, dehydroemetine, diethylcarbamazine,diloxanide furoate, eflornithine, furazolidone, glucocorticoids,halofantrine, iodoquinol, ivermectin, mebendazole, mefloquine, meglumineantimoniate, melarsoprol, metrifonate, metronidazole, niclosamide,nifurtimox, oxamniquine, paromomycin, pentamidine isethionate,piperazine, praziquantel, primaquine phosphate, proguanil, pyrantelpamoate, pyrimethanmine-sulfonamides, pyrimethanmine-sulfadoxine,quinacrine HC, quinine sulfate, quinidine gluconate, spiramycin,stibogluconate sodium (sodium antimony gluconate), suramin,tetracycline, doxycycline, thiabendazole, tinidazole,trimethroprim-sulfamethoxazole, and tryparsamide some of which are usedalone or in combination with others.

Antibacterial agents kill or inhibit the growth or function of bacteria.A large class of antibacterial agents is antibiotics. Antibiotics, whichare effective for killing or inhibiting a wide range of bacteria, arereferred to as broad spectrum antibiotics. Other types of antibioticsare predominantly effective against the bacteria of the classgram-positive or gram-negative. These types of antibiotics are referredto as narrow spectrum antibiotics. Other antibiotics which are effectiveagainst a single organism or disease and not against other types ofbacteria, are referred to as limited spectrum antibiotics. Antibacterialagents are sometimes classified based on their primary mode of action.In general, antibacterial agents are cell wall synthesis inhibitors,cell membrane inhibitors, protein synthesis inhibitors, nucleic acidsynthesis or functional inhibitors, and competitive inhibitors.

Antiviral agents are compounds which prevent infection of cells byviruses or replication of the virus within the cell. There are manyfewer antiviral drugs than antibacterial drugs because the process ofviral replication is so closely related to DNA replication within thehost cell, that non-specific antiviral agents would often be toxic tothe host. There are several stages within the process of viral infectionwhich can be blocked or inhibited by antiviral agents. These stagesinclude, attachment of the virus to the host cell (immunoglobulin orbinding peptides), uncoating of the virus (e.g. amantadine), synthesisor translation of viral mRNA (e.g. interferon), replication of viral RNAor DNA (e.g. nucleotide analogs), maturation of new virus proteins (e.g.protease inhibitors), and budding and release of the virus.

Nucleotide analogs are synthetic compounds which are similar tonucleotides, but which have an incomplete or abnormal deoxyribose orribose group. Once the nucleotide analogs are in the cell, they arephosphorylated, producing the triphosphate formed which competes withnormal nucleotides for incorporation into the viral DNA or RNA. Once thetriphosphate form of the nucleotide analog is incorporated into thegrowing nucleic acid chain, it causes irreversible association with theviral polymerase and thus chain termination. Nucleotide analogs include,but are not limited to, acyclovir (used for the treatment of herpessimplex virus and varicella-zoster virus), gancyclovir (useful for thetreatment of cytomegalovirus), idoxuridine, ribavirin (useful for thetreatment of respiratory syncitial virus), dideoxyinosine,dideoxycytidine, zidovudine (azidothymidine), imiquimod, andresimiquimod.

The interferons are cytokines which are secreted by virus-infected cellsas well as immune cells. The interferons function by binding to specificreceptors on cells adjacent to the infected cells, causing the change inthe cell which protects it from infection by the virus. α andβ-interferon also induce the expression of Class I and Class II MHCmolecules on the surface of infected cells, resulting in increasedantigen presentation for host immune cell recognition. α andβ-interferons are available a recombinant forms and have been used forthe treatment of chronic hepatitis B and C infection. At the dosageswhich are effective for anti-viral therapy, interferons have severe sideeffects such as fever, malaise and weight loss.

Anti-viral agents useful in the invention include but are not limited toimmunoglobulins, amantadine, interferons, nucleotide analogs, andprotease inhibitors. Specific examples of anti-virals include but arenot limited to Acemannan; Acyclovir; Acyclovir Sodium; Adefovir;Alovudine; Alvircept Sudotox; Amantadine Hydrochloride; Aranotin;Arildone; Atevirdine Mesylate; Avridine; Cidofovir, Cipamnfylline;Cytarabine Hydrochloride; Delavirdine Mesylate; Desciclovir, Didanosine;Disoxaril; Edoxudine; Enviradene; Enviroxime; Famciclovir; FamotineHydrochloride; Fiacitabine; Fialuridine; Fosarilate; Foscarnet Sodium;Fosfonet Sodium; Ganciclovir; Ganciclovir Sodium; Idoxuridine; Kethoxal;Lamivudine; Lobucavir; Memotine Hydrochloride; Methisazone; Nevirapine;Penciclovir; Pirodavir; Ribavirin; Rimantadine Hydrochloride; SaquinavirMesylate; Somantadine Hydrochloride; Sorivudine; Statolon; Stavudine;Tilorone Hydrochloride; Trifluridine; Valacyclovir Hydrochloride;Vidarabine; Vidarabine Phosphate, Vidarabine Sodium Phosphate; Viroxime;Zalcitabine; Zidovudine; and Zinviroxime.

Anti-fungal agents are useful for the treatment and prevention ofinfective fungi. Anti-fungal agents are sometimes classified by theirmechanism of action. Some anti-fungal agents function a cell wallinhibitors by inhibiting glucose synthase. These include, but are notlimited to, basiungin/ECB. Other anti-fungal agents function bydestabilizing membrane integrity. These include, but are not limited to,immidazoles, such as clotrimazole, sertaconzole, fluconazole,itraconazole, ketoconazole, miconazole, and voriconacole, as well as FK463, amphotericin B, BAY 38-9502, MK 991, pradimicin, UK 292,butenafine, and terbinafine. Other anti-fungal agents function bybreaking down chitin (e.g. chitinase) or immunosuppression (501 cream).

CpG immunostimulatory oligonucleotides can be combined with othertherapeutic agents such as adjuvants to enhance immune responses. TheCpG immunostimulatory oligonucleotide and other therapeutic agent may beadministered simultaneously or sequentially. When the other therapeuticagents are administered simultaneously they can be administered in thesame or separate formulations, but are administered at the same time.The other therapeutic agents are administered sequentially with oneanother and with CpG immunostimulatory oligonucleotide, when theadministration of the other therapeutic agents and the CpGimmunostimulatory oligonucleotide is temporally separated. Theseparation in time between the administration of these compounds may bea matter of minutes or it may be longer. Other therapeutic agentsinclude but are not limited to adjuvants, cytokines, antibodies,antigens, etc.

The compositions of the invention may also be administered withnon-nucleic acid adjuvants. A non-nucleic acid adjuvant is any moleculeor compound except for the CpG immunostimulatory oligonucleotidesdescribed herein which can stimulate the humoral and/or cellular immuneresponse. Non-nucleic acid adjuvants include, for instance, adjuvantsthat create a depo effect, immune stimulating adjuvants, and adjuvantsthat create a depo effect and stimulate the immune system.

The CpG immunostimulatory oligonucleotides are also useful as mucosaladjuvants. It has previously been discovered that both systemic andmucosal immunity are induced by mucosal delivery of CpG nucleic acids.Thus, the oligonucleotides may be administered in combination with othermucosal adjuvants.

Immune responses can also be induced or augmented by theco-administration or co-linear expression of cytokines (Bueler &Mulligan, 1996; Chow et al., 1997; Geissler et al., 1997; Iwasaki etal., 1997; Kim et al., 1997) or B-7 co-stimulatory molecules (Iwasai etal., 1997; Tsuji et al., 1997) with the CpG immunostimulatoryoligonucleotides. The term cytokine is used as a generic name for adiverse group of soluble proteins and peptides which act as humoralregulators at nano- to picomolar concentrations and which, either undernormal or pathological conditions, modulate the functional activities ofindividual cells and tissues. These proteins also mediate interactionsbetween cells directly and regulate processes taking place in theextracellular environment. Examples of cytokines include, but are notlimited to IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15,IL-18, granulocyte-macrophage colony stimulating factor (GM-CSF),granulocyte colony stimulating factor (G-CSF), interferon-γ (γ-IFN),IFN-α, tumor necrosis factor (TNF), TGF-β, FLT-3 ligand, and CD40ligand.

The oligonucleotides are also useful for redirecting an immune responsefrom a Th2 immune response to a Th1 immune response. This results in theproduction of a relatively balanced Th1/Th2 environment. Redirection ofan immune response from a Th2 to a Th1 immune response can be assessedby measuring the levels of cytokines produced in response to the nucleicacid (e.g., by inducing monocytic cells and other cells to produce Th1cytokines, including IL-12, IFN-γ and GM-CSF). The redirection orrebalance of the immune response from a Th2 to a Th1 response isparticularly useful for the treatment or prevention of asthma. Forinstance, an effective amount for treating asthma can be that amount;useful for redirecting a Th2 type of immune response that is associatedwith asthma to a Th1 type of response or a balanced Th1/Th2 environment.Th2 cytokines, especially IL-4 and IL-5 are elevated in the airways ofasthmatic subjects. The CpG immunostimulatory oligonucleotides of theinvention cause an increase in Th1 cytokines which helps to rebalancethe immune system, preventing or reducing the adverse effects associatedwith a predominately Th2 immune response.

The oligonucleotides of the invention may also be useful for treatingairway remodeling. Airway remodeling results from smooth muscle cellproliferation and/or submucosal thickening in the airways, andultimately causes narrowing of the airways leading to restrictedairflow. The oligonucleotides of the invention may prevent furtherremodeling and possibly even reduce tissue build up resulting from theremodeling process.

The oligonucleotides are also useful for improving survival,differentiation, activation and maturation of dendritic cells. The CpGimmunostimulatory oligonucleotides have the unique capability to promotecell survival, differentiation, activation and maturation of dendriticcells.

CpG immunostimulatory oligonucleotides also increase natural killer celllytic activity and antibody dependent cellular cytotoxicity (ADCC). ADCCcan be performed using a CpG immunostimulatory oligonucleotide incombination with an antibody specific for a cellular target, such as acancer cell. When the CpG immunostimulatory oligonucleotide isadministered to a subject in conjunction with the antibody the subject'simmune system is induced to kill the tumor cell. The antibodies usefulin the ADCC procedure include antibodies which interact with a cell inthe body. Many such antibodies specific for cellular targets have beendescribed in the art and many are commercially available.

The CpG immunostimulatory oligonucleotides may also be administered inconjunction with an anti-cancer therapy. Anti-cancer therapies includecancer medicaments, radiation and surgical procedures. As used herein, a“cancer medicament” refers to a agent which is administered to a subjectfor the purpose of treating a cancer. As used herein, “treating cancer”includes preventing the development of a cancer, reducing the symptomsof cancer, and/or inhibiting the growth of an established cancer. Inother aspects, the cancer medicament is administered to a subject atrisk of developing a cancer for the purpose of reducing the risk ofdeveloping the cancer. Various types of medicaments for the treatment ofcancer are described herein. For the purpose of this specification,cancer medicaments are classified as chemotherapeutic agents,immunotherapeutic agents, cancer vaccines, hormone therapy, andbiological response modifiers.

Additionally, the methods of the invention are intended to embrace theuse of more than one cancer medicament along with the CpGimmunostimulatory oligonucleotides. As an example, where appropriate,the CpG immunostimulatory oligonucleotides may be administered with botha chemotherapeutic agent and an immunotherapeutic agent. Alternatively,the cancer medicament may embrace an immunotherapeutic agent and acancer vaccine, or a chemotherapeutic agent and a cancer vaccine, or achemotherapeutic agent, an immunotherapeutic agent and a cancer vaccineall administered to one subject for the purpose of treating a subjecthaving a cancer or at risk of developing a cancer.

The chemotherapeutic agent may be selected from the group consisting ofmethotrexate, vincristine, adriamycin, cisplatin, non-sugar containingchloroethylnitrosoureas, 5-fluorouracil, mitomycin C, bleomycin,doxorubicin, dacarbazine, taxol, fragyline, Meglamine GLA, valrubicin,carmustaine and poliferposan, MMI270, BAY 12-9566, RAS farnesyltransferase inhibitor, farnesyl transferase inhibitor, MMP,MTA/LY231514, LY264618/Lometexol, Glamolec, CI-994, TNP-470,Hycamtin/Topotecan, PKC412, Valspodar/PSC833, Novantrone/Mitroxantrone,Metaret/Suramin, Batimastat, E7070, BCH-4556, CS-682, 9-AC, AG3340,AG3433, Incel/VX-710, VX-853, ZD0101, ISI641, ODN 698, TA2516/Marmistat, BB2516/Marmistat, CDP 845, D2163, PD183805, DX8951f,Lemonal DP 2202, FK 317, Picibanil/OK-432, AD 32/Valrubicin,Metastron/strontium derivative, Temodal/Temozolomide, Evacet/liposomaldoxorubicin, Yewtaxan/Paclitaxel, Taxol/Paclitaxel, Xeload/Capecitabine,Furtulon/Doxifluridine, Cyclopax/oral paclitaxel, Oral Taxoid,SPU-077/Cisplatin, HMR 1275/Flavopiridol, CP-358 (774)/BGFR, CP-609(754)/RAS oncogene inhibitor, BMS-182751/oral platinum,UFT(Tegafur/Uracil), Ergamisol/Levamisole, Eniluracil/776C85/5FUenhancer, Campto/Levamisole, Camptosar/Irinotecan, Turnodex/Ralitrexed,Leustatin/Cladribine, Paxex/Paclitaxel, Doxil/liposomal doxorubicin,Caelyz/liposomal doxorubicin, Fludara/Fludarabine,Pharmarubicin/Epirubicin, DepoCyt, ZD1839, LU 79553/Bis-Naphtalimide, LU103793/Dolastain, Caetyx/liposomal doxorubicin, Gemzar/Gemcitabine, ZD0473/Anormed, YM 116, Iodine seeds, CDK4 and CDK2 inhibitors, PARPinhibitors, D4809/Dexifosamide, Ifes/Mesnex/Ifosamide, Vumon/Teniposide,Paraplatin/Carboplatin, Plantinol/cisplatin, Vepeside/Etoposide, ZD9331, Taxotere/Docetaxel, prodrug of guanine arabinoside, Taxane Analog,nitrosoureas, alkylating agents such as melphelan and cyclophosphamide,Aminoglutethimide, Asparaginase, Busulfan, Carboplatin, Chlorombucil,Cytarabine HCl, Dactinomycin, Daunorubicin HCl, Estramustine phosphatesodium, Etoposide (VP16-213), Floxuridine, Fluorouracil (5-FU),Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide, InterferonAlfa-2a, Alfa-2b, Leuprolide acetate (LHRH-releasing factor analog),Lomustine (CCNU), Mechlorethamine HCl (nitrogen mustard),Mercaptopurine, Mesna, Mitotane (o,p′-DDD), Mitoxantrone HCl,Octreotide, Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifencitrate, Thioguanine, Thiotepa, Vinblastine sulfate, Amsacrine (m-AMSA),Azacitidine, Erthropoietin, Hexamethylmelamine (HMM), Interleukin 2,Mitoguazone (methyl-GAG; methyl glyoxal bis-guanylhydrazone; MGBG),Pentostatin (2′deoxycoformycin), Semustine (methyl-CCNU), Teniposide(VM-26) and Vindesine sulfate, but it is not so limited.

The immunotherapeutic agent may be selected from the group consisting ofRibutaxin, Herceptin, Quadramet, Panorex, IDEC-Y2B8, BEC2, C225,Oncolym, SMART M195, ATRAGEN, Ovarex, Bexxar, LDP-03, ior t6, MDX-210,MDX-11, MDX-22, OV103, 3622 W94, anti-VEGF, Zenapax, MDX-220, MDX-447,MELIMMUNE-2, MELIMMUNE-1, CEACIDE, Pretarget, NovoMAb-G2, TNT,Gliomab-H, GNI-250, EMD-72000, LymphoCide, CMA 676, Monopharm-C, 4B5,ior egf.r3, ior c5, BABS, anti-FLK-2, MDX-260, ANA Ab, SMART 1D10 Ab,SMART ABL 364 Ab and ImmuRAIT-CEA, but it is not so limited.

The cancer vaccine may be selected from the group consisting of EGF,Anti-idiotypic cancer vaccines, Gp75 antigen. GMK melanoma vaccine, MGVganglioside conjugate vaccine, Her2/neu, Ovarex, M-Vax, O-Vax, L-Vax,STn-KHL theratope, BLP25 (MUC-1), liposomal idiotypic vaccine, Melacine,peptide antigen vaccines, toxin/antigen vaccines, MVA-based vaccine,PACIS, BCG vacine, TA-HPV, TA-CIN, DISC-virus and ImmuCyst/TheraCys, butit is not so limited.

The use of CpG immunostimulatory oligonucleotides in conjunction withimmunotherapeutic agents such as monoclonal antibodies is able toincrease long-term survival through a number of mechanisms includingsignificant enhancement of ADCC (as discussed above), activation ofnatural killer (NK) cells and an increase in IFNα levels. The nucleicacids when used in combination with monoclonal antibodies serve toreduce the dose of the antibody required to achieve a biological result.

As used herein, the terms “cancer antigen” and “tumor antigen” are usedinterchangeably to refer to antigens which are differentially expressedby cancer cells and can thereby be exploited in order to target cancercells. Cancer antigens are antigens which can potentially stimulateapparently tumor-specific immune responses. Some of these antigens areencoded, although not necessarily expressed, by normal cells. Theseantigens can be characterized as those which are normally silent (i.e.,not expressed) in normal cells, those that are expressed only at certainstages of differentiation and those that are temporally expressed suchas embryonic and fetal antigens. Other cancer antigens are encoded bymutant cellular genes, such as oncogenes (e.g., activated as oncogene),suppressor genes (e.g., mutant p53), fusion proteins resulting frominternal deletions or chromosomal translocations. Still other cancerantigens can be encoded by viral genes such as those carried on RNA andDNA tumor viruses.

The CpG immunostimulatory oligonucleotides are also useful for treatingand preventing autoimmune disease. Autoimmune disease is a class ofdiseases in which an subject's own antibodies react with host tissue orin which immune effector T cells are autoreactive to endogenous selfpeptides and cause destruction of tissue. Thus an immune response ismounted against a subject's own antigens, referred to as self antigens.Autoimmune diseases include but are not limited to rheumatoid arthritis,Crohn's disease, multiple sclerosis, systemic lupus erythematosus (SLE),autoimmune encephalomyelitis, myasthenia gravis (MG), Hashimoto'sthyroiditis, Goodpasture's syndrome, pemphigus (e.g., pemphigusvulgaris), Grave's disease, autoimmune hemolytic anemia, autoimmunethrombocytopenic purpura, scleroderma with anti-collagen antibodies,mixed connective tissue disease, polymyositis, pernicious anemia,idiopathic Addison's disease, autoimmune-associated infertility,glomerulonephritis (e.g., crescentic glomerulonephritis, proliferativeglomerulonephritis), bullous pemphigoid. Sjögren's syndrome, insulinresistance, and autoimmune diabetes mellitus.

A “self-antigen” as used herein refers to an antigen of a normal hosttissue. Normal host tissue does not include cancer cells. Thus an immuneresponse mounted against a self-antigen, in the context of an autoimmunedisease, is an undesirable immune response and contributes todestruction and damage of normal tissue, whereas an immune responsemounted against a cancer antigen is a desirable immune response andcontributes to the destruction of the tumor or cancer. Thus, in someaspects of the invention aimed at treating autoimmune disorders it isnot recommended that the CpG immunostimulatory nucleic acids beadministered with self antigens, particularly those that are the targetsof the autoimmune disorder.

In other instances, the CpG immunostimulatory nucleic acids may bedelivered with low doses of self-antigens. A number of animal studieshave demonstrated that mucosal administration of low doses of antigencan result in a state of immune hyporesponsiveness or “tolerance.” Theactive mechanism appears to be a cytokine-mediated immune deviation awayfrom a Th1 towards a predominantly Th2 and Th3 (i.e., TGF-β dominated)response. The active suppression with low dose antigen delivery can alsosuppress an unrelated immune response (bystander suppression) which isof considerable interest in the therapy of autoimmune diseases, forexample, rheumatoid arthritis and SLE. Bystander suppression involvesthe secretion of Th1-counter-regulatory, suppressor cytokines in thelocal environment where proinflammatory and Th1 cytokines are releasedin either an antigen-specific or antigen-nonspecific manner. “Tolerance”as used herein is used to refer to this phenomenon. Indeed, oraltolerance has been effective in the treatment of a number of autoimmunediseases in animals including: experimental autoimmune encephalomyelitis(EAE), experimental autoimmune myasthenia gravis, collagen-inducedarthritis (CIA), and insulin-dependent diabetes mellitus. In thesemodels, the prevention and suppression of autoimmune disease isassociated with a shift in antigen-specific humoral and cellularresponses from a Th1 to Th2/Th3 response.

The invention also includes a method for inducing antigen non-specificinnate immune activation and broad spectrum resistance to infectiouschallenge using the CpG immunostimulatory oligonucleotides. The termantigen non-specific innate immune activation as used herein refers tothe activation of immune cells other than B cells and for instance caninclude the activation of NK cells, T cells or other immune cells thatcan respond in an antigen independent fashion or some combination ofthese cells. A broad spectrum resistance to infectious challenge isinduced because the immune cells are in active form and are primed torespond to any invading compound or microorganism. The cells do not haveto be specifically primed against a particular antigen. This isparticularly useful in biowarfare, and the other circumstances describedabove such as travelers.

The CpG immunostimulatory oligonucleotides may be directly administeredto the subject or may be administered in conjunction with a nucleic aciddelivery complex. A nucleic acid delivery complex shall mean a nucleicacid molecule associated with (e.g. ionically or covalently bound to; orencapsulated within) a targeting means (e.g. a molecule that results inhigher affinity binding to target cell. Examples of nucleic aciddelivery complexes include nucleic acids associated with a sterol (e.g.cholesterol), a lipid (e.g. a cationic lipid, virosome or liposome), ora target cell specific binding agent (e.g. a ligand recognized by targetcell specific receptor). Preferred complexes may be sufficiently stablein vivo to prevent significant uncoupling prior to internalization bythe target cell. However, the complex can be cleavable under appropriateconditions within the cell so that the oligonucleotide is released in afunctional form.

Delivery vehicles or delivery devices for delivering antigen andoligonucleotides to surfaces have been described. The CpGimmunostimulatory oligonucleotide and/or the antigen and/or othertherapeutics may be administered alone (e.g., in saline or buffer) orusing any delivery vehicles known in the art. For instance the followingdelivery vehicles have been described: Cochleates; Emulsomes, ISCOMs;Liposomes; Live bacterial vectors (e.g., Salmonella, Escherichia coli,Bacillus calmatte-guerin, Shigella, Lactobacillus); Live viral vectors(e.g., Vaccinia, adenovirus, Herpes Simplex); Microspheres; Nucleic acidvaccines; Polymers; Polymer rings; Proteosomes; Sodium Fluoride;Transgenic plants; Virosomes; Virus-like particles. Other deliveryvehicles are known in the art and some additional examples are providedbelow in the discussion of vectors.

The term effective amount of a CpG immunostimulatory oligonucleotiderefers to the amount necessary or sufficient to realize a desiredbiologic effect. For example, an effective amount of a CpGimmunostimulatory oligonucleotide administered with an antigen forinducing mucosal immunity is that amount necessary to cause thedevelopment of IgA in response to an antigen upon exposure to theantigen, whereas that amount required for inducing systemic immunity isthat amount necessary to cause the development of IgG in response to anantigen upon exposure to the antigen. Combined with the teachingsprovided herein, by choosing among the various active compounds andweighing factors such as potency, relative bioavailability, patient bodyweight, severity of adverse side-effects and preferred mode ofadministration, an effective prophylactic or therapeutic treatmentregimen can be planned which does not cause substantial toxicity and yetis entirely effective to treat the particular subject. The effectiveamount for any particular application can vary depending on such factorsas the disease or condition being treated, the particular CpGimmunostimulatory oligonucleotide being administered the size of thesubject, or the severity of the disease or condition. One of ordinaryskill in the art can empirically determine the effective amount of aparticular CpG immunostimulatory olignucleotide and/or antigen and/orother therapeutic agent without necessitating undue experimentation.

Subject doses of the compounds described herein for mucosal or localdelivery typically range from about 0.1 g to 10 mg per administration,which depending on the application could be given daily, weekly, ormonthly and any other amount of time therebetween. More typicallymucosal or local doses range from about 10 μg to 5 mg peradministration, and most typically from about 100 μg to 1 mg, with 2-4administrations being spaced days or weeks apart. More typically, immunestimulant doses range from 1 μg to 10 mg per administration, and mosttypically 10 μg to 1 mg, with daily or weekly administrations. Subjectdoses of the compounds described herein for parenteral delivery for thepurpose of inducing an antigen-specific immune response, wherein thecompounds are delivered with an antigen but not another therapeuticagent are typically 5 to 10,000 times higher than the effective mucosaldose for vaccine adjuvant or immune stimulant applications, and moretypically 10 to 1,000 times higher, and most typically 20 to 100 timeshigher. Doses of the compounds described herein for parenteral deliveryfor the purpose of inducing an innate immune response or for increasingADCC or for inducing an antigen specific immune response when the CpGimmunostimulatory oligonucleotides are administered in combination withother therapeutic agents or in specialized delivery vehicles typicallyrange from about 0.1 μg to 10 mg per administration, which depending onthe application could be given daily, weekly, or monthly and any otheramount of time therebetween. More typically parenteral doses for thesepurposes range from about 10 μg to 5 mg per administration, and mosttypically from about 100 μg to 1 mg, with 2-4 administrations beingspaced days or weeks apart. In some embodiments, however, parenteraldoses for these purposes may be used in a range of 5 to 10,000 timeshigher than the typical doses described above.

For any compound described herein the therapeutically effective amountcan be initially determined from animal models. A therapeuticallyeffective dose can also be determined from human data for CpGoligonucleotides which have been tested in humans (human clinical trialshave been initiated) and for compounds which are known to exhibitsimilar pharmacological activities, such as other adjuvants, e.g., LTand other antigens for vaccination purposes. Higher doses may berequired for parenteral administration. The applied dose can be adjustedbased on the relative bioavailability and potency of the administeredcompound. Adjusting the dose to achieve maximal efficacy based on themethods described above and other methods as are well-known in the artis well within the capabilities of the ordinarily skilled artisan.

The formulations of the invention are administered in pharmaceuticallyacceptable solutions, which may routinely contain pharmaceuticallyacceptable concentrations of salt, buffering agents, preservatives,compatible carriers, adjuvants, and optionally other therapeuticingredients.

For use in therapy, an effective amount of the CpG immunostimulatoryoligonucleotide can be administered to a subject by any mode thatdelivers the oligonucleotide to the desired surface, e.g., mucosal,systemic. Administering the pharmaceutical composition of the presentinvention may be accomplished by any means known to the skilled artisan.Preferred routes of administration include but are not limited to oral,parenteral, intramuscular, intranasal, sublingual, intratracheal,inhalation, ocular, vaginal, and rectal.

For oral administration, the compounds (i.e., CpG immunostimulatoryoligonucleotides, antigens and other therapeutic agents) can beformulated readily by combining the active compound(s) withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the compounds of the invention to be formulated astablets, pills, dragees capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a subject to be treated.Pharmaceutical preparations for oral use can be obtained as solidexcipient, optionally grinding a resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cares. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate. Optionally the oral formulations may also be formulated insaline or buffers, i.e. EDTA for neutralizing internal acid conditionsor may be administered without any carriers.

Also specifically contemplated are oral dosage forms of the abovecomponent or components. The component or components may be chemicallymodified so that oral delivery of the derivative is efficacious.Generally, the chemical modification contemplated is the attachment ofat least one moiety to the component molecule itself where said moietypermits (a) inhibition of proteolysis; and (b) uptake into the bloodstream from the stomach or intestine. Also desired is the increase inoverall stability of the component or components and increase incirculation time in the body. Examples of such moieties include:polyethylene glycol, copolymers of ethylene glycol and propylene glycol,carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone and polyproline. Abuchowski and Davis, 1981, “SolublePolymer-Enzyme Adducts” In: Enzymes as Drugs, Hocenberg and Roberts,eds., Wiley-Interscience, New York, N.Y., pp. 367-383; Newmark, et al.,1982, J. Appl. Biochem. 4:185-189. Other polymers that could be used arepoly-1,3-dioxolan and poly-1,3,6-tioxocane. Preferred for pharmaceuticalusage, as indicated above, are polyethylene glycol moieties.

For the component (or derivative) the location of release may be thestomach, the small intestine (the duodenum, the jejunum, or the ileum),or the large intestine. One skilled in the art has availableformulations which will not dissolve in the stomach, yet will releasethe material in the duodenum or elsewhere in the intestine. Preferably,the release will avoid the deleterious effects of the stomachenvironment, either by protection of the oligonucleotide (or derivative)or by release of the biologically active material beyond the stomachenvironment, such as in the intestine.

To ensure full gastric resistance a coating impermeable to at least pH5.0 is essential. Examples of the more common inert ingredients that areused as enteric coatings are cellulose acetate trimellitate (CAT),hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55,polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, celluloseacetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. Thesecoatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which arenot intended for protection against the stomach. This can include sugarcoatings, or coatings which make the tablet easier to swallow. Capsulesmay consist of a hard shell (such as gelatin) for delivery of drytherapeutic i.e. powder, for liquid forms, a soft gelatin shell may beused. The shell material of cachets could be thick starch or otheredible paper. For pills, lozenges, molded tablets or tablet triturates,moist massing techniques can be used.

The therapeutic can be included in the formulation as finemulti-particulates in the form of granules or pellets of particle sizeabout 1 nm. The formulation of the material for capsule administrationcould also be as a powder, lightly compressed plugs or even as tablets.The therapeutic could be prepared by compression.

Colorants and flavoring agents may all be included. For example, theoligonucleotide (or derivative) may be formulated (such as by liposomeor microsphere encapsulation) and then further contained within anedible product, such as a refrigerated beverage containing colorants andflavoring agents.

One may dilute or increase the volume of the therapeutic with an inertmaterial. These diluents could include carbohydrates, especiallymannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modifieddextrans and starch. Certain inorganic salts may be also be used asfillers including calcium triphosphate magnesium carbonate and sodiumchloride. Some commercially available diluents are Fast-Flo, Emdex,STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic intoa solid dosage form. Materials used as disintegrates include but are notlimited to starch, including the commercial disintegrant based onstarch, Explotab. Sodium starch glycolate, Amberlite, sodiumcarboxymethylcellulose, ultramylopectin, sodium alginate, gelatin,orange peel, acid carboxymethyl cellulose, natural sponge and bentonitemay all be used. Another form of the disintegrants are the insolublecationic exchange resins. Powdered gums may be used as disintegrants andas binders and these can include powdered gums such as agar, Karaya ortragacanth. Alginic acid and its sodium salt are also useful asdisintegrants.

Binders may be used to hold the therapeutic agent together to form ahard tablet and include materials from natural products such as acacia,tragacanth, starch and gelatin. Other include methyl cellulose (MC),ethyl cellulose (BC) and carboxymethyl cellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both beused in alcoholic solutions to granulate the therapeutic.

An anti-frictional agent may be included in the formulation of thetherapeutic to prevent sticking during the formulation process.Lubricants may be used as a layer between the therapeutic and the diewall, and these can include but are not limited to; stearic acidincluding its magnesium and calcium salts, polytetrafluoroethylene(PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricantsmay also be used such as sodium lauryl sulfate, magnesium laurylsulfate, polyethylene glycol of various molecular weights, Carbowax 4000and 6000.

Glidants that might improve the flow properties of the drug duringformulation and to aid rearrangement during compression might be added.The glidants may include starch, talc, pyrogenic silica and hydratedsilicoaluminate.

To aid dissolution of the therapeutic into the aqueous environment asurfactant might be added as a wetting agent. Surfactants may includeanionic detergents such as sodium lauryl sulfate, dioctyl sodiumsulfosuccinate and dioctyl sodium sulfonate. Cationic detergents mightbe used and could include benzalkonium chloride or benzethomiumchloride. The list of potential nonionic detergents that could beincluded in the formulation as surfactants are lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and60, glycerol monosterate, polysorbate 40, 60, 65 and 80, sucrose fattyacid ester, methyl cellulose and carboxymethyl cellulose. Thesesurfactants could be present in the formulation of the oligonucleotideor derivative either alone or as a mixture in different ratios.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. Microspheres formulatedfor oral administration may also be used. Such microspheres have beenwell defined in the art. All formulations for oral administration shouldbe in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention may be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

Also contemplated herein is pulmonary delivery of the oligonucleotides(or derivatives thereof). The oligonucleotide (or derivative) isdelivered to the lungs of a mammal while inhaling and traverses acrossthe lung epithelial lining to the blood stream. Other reports of inhaledmolecules include Adjei et al., 1990, Pharmaceutical Research,7:565-569; Adjei et al., 1990, International Journal of Pharmaceutics,63:135-144 (leuprolide acetate); Braquet et al., 1989, Journal ofCardiovascular Pharmacology, 13(suppl. 5):143-146 (endothelin-1);Hubbard et al., 1989, Annals of Internal Medicine, Vol. III, pp. 206-212(al-antitrypsin); Smith et al., 1989, J. Clin. Invest. 84:1145-1146(a-1-proteinase); Oswein et al., 1990, “Aeosolization of Proteins”,Proceedings of Symposium on Respiratory Drug Delivery II, Keystone,Colo., March, (recombinant human growth hormone); Debs et al., 1988, J.Immunol. 140:3482-3488 (interferon-g and tumor necrosis factor alpha)and Platz et al, U.S. Pat. No. 5,284,656 (granulocyte colony stimulatingactor). A method and composition for pulmonary delivery of drugs forsystemic effect is described in U.S. Pat. No. 5,451,569, issued Sep. 19,1995 to Wong et al.

Contemplated for use in the practice of this invention are a wide rangeof mechanical devices designed for pulmonary delivery of therapeuticproducts, including but not limited to nebulizers, metered dose inhales,and powder inhalers, all of which are familiar to those skilled in theart.

Some specific examples of commercially available devices suitable forthe practice of this invention are the Ultravent nebulizer, manufacturedby Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer,manufactured by Marquest Medical Products, Englewood, Colo.; theVentolin metered dose inhaler, manufactured by Glaxo Inc., ResearchTriangle Park, North Carolina; and the Spinhaler powder inhaler,manufactured by Fisons Corp., Bedford, Mass.

All such devices require the use of formulations suitable for thedispensing of oligonucleotide (or derivative). Typically, eachformulation is specific to the type of device employed and may involvethe use of an appropriate propellant material, in addition to the usualdiluents, adjuvants and/or carriers useful in therapy. Also, the use ofliposomes, microcapsules or microspheres, inclusion complexes, or othertypes of carriers is contemplated. Chemically modified oligonucleotidemay also be prepared in different formulations depending on the type ofchemical modification or the type of device employed.

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise oligonucleotide (or derivative)dissolved in water at a concentration of about 0.1 to 25 mg ofbiologically active oligonucleotide per mL of solution. The formulationmay also include a buffer and a simple sugar (e.g., for oligonucleotidestabilization and regulation of osmotic pressure). The nebulizerformulation may also contain a surfactant, to reduce or prevent surfaceinduced aggregation of the oligonucleotide caused by atomization of thesolution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the oligonucleotide (orderivative) suspended in a propellant with the aid of a surfactant. Thepropellant may be any conventional material employed for this purpose,such as a chlorofluorocarbon, a hydrochlorofluorocarbon, ahydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane,dichlorodifluoromethane, di and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing oligonucleotide (or derivative) andmay also include a bulking agent such as lactose, sorbitol, sucrose, ormannitol in amounts which facilitate dispersal of the powder from thedevice, e.g., 50 to 90% by weight of the formulation. Theoligonucleotide (or derivative) should most advantageously be preparedin particulate form with an average particle size of less than 10 mm (ormicrons), most preferably 0.5 to 5 mm, for most effective delivery tothe distal lung.

Nasal delivery of a pharmaceutical composition of the present inventionis also contemplated. Nasal delivery allows the passage of apharmaceutical composition of the present invention to the blood streamdirectly after administering the therapeutic product to the nose,without the necessity for deposition of the product in the lung.Formulations for nasal delivery include those with dextran orcyclodextran.

For nasal administration, a useful device is a small, hard bottle towhich a metered dose sprayer is attached. In one embodiment, the metereddose is delivered by drawing the pharmaceutical composition of thepresent invention solution into a chamber of defined volume, whichchamber has an aperture dimensioned to aerosolize and aerosolformulation by forming a spray when a liquid in the chamber iscompressed. The chamber is compressed to administer the pharmaceuticalcomposition of the present invention. In a specific embodiment, thechamber is a piston arrangement. Such devices are commerciallyavailable.

Alternatively, a plastic squeeze bottle with an aperture or openingdimensioned to aerosolize an aerosol formulation by forming a spray whensqueezed is used. The opening is usually found in the top of the bottle,and the top is generally tapered to partially fit in the nasal passagesfor efficient administration of the aerosol formulation. Preferably, thenasal inhaler will provide a metered amount of the aerosol formulation,for administration of a measured dose of the drug.

The compounds, when it is desirable to deliver them systemically, may beformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active compounds may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal or vaginal compositionssuch as suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Suitable liquid or solid pharmaceutical preparation forms are, forexample, aqueous or saline solutions for inhalation, microencapsulated,encochleated, coated onto microscopic gold particles, contained inliposomes, nebulized, aerosols, pellets for implantation into the skin,or dried onto a sharp object to be scratched into the skin. Thepharmaceutical compositions also include granules, powders, tablets,coated tablets, (micro)capsules, suppositories, syrups, emulsions,suspensions, creams, drops or preparations with protracted release ofactive compounds, in whose preparation excipients and additives and/orauxiliaries such as disintegrants, binders, coating agents, swellingagents lubricants, flavorings, sweeteners or solubilizers arecustomarily used as described above. The pharmaceutical compositions aresuitable for use in a variety of drug delivery systems. For a briefreview of methods for drug delivery, see Langer, Science 249:1527-1533,1990, which is incorporated herein by reference.

The CpG immunostimulatory oligonucleotides and optionally othertherapeutics and/or antigens may be administered per se (neat) or in theform of a pharmaceutically acceptable salt. When used in medicine thesalts should be pharmaceutically acceptable, but non-pharmaceuticallyacceptable salts may conveniently be used to prepare pharmaceuticallyacceptable salts thereof. Such salts include, but are not limited to,those prepared from the following acids: hydrochloric, hydrobromic,sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluenesulphonic, tartaric, citric, methane sulphonic, formic, malonic,succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also, suchsalts can be prepared as alkaline metal or alkaline earth salts, such assodium, potassium or calcium salts of the carboxylic acid group.

Suitable buffering agents include: acetic acid and a salt (1-2% w/v);citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v);and phosphoric acid and a salt (0.8-2% w/v). Suitable preservativesinclude benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9%w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).

The pharmaceutical compositions of the invention contain an effectiveamount of a CpG immunostimulatory oligonucleotide and optionallyantigens and/or other therapeutic agents optionally included in apharmaceutically-acceptable carrier. The termpharmaceutically-acceptable carrier means one or more compatible solidor liquid filler, diluents or encapsulating substances which aresuitable for administration to a human or other vertebrate animal. Theterm carrier denotes an organic or inorganic ingredient, natural orsynthetic, with which the active ingredient is combined to facilitatethe application. The components of the pharmaceutical compositions alsoare capable of being commingled with the compounds of the presentinvention, and with each other, in a manner such that there is nointeraction which would substantially impair the desired pharmaceuticalefficiency.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLES Materials and Methods

Oligodeoxynucleotides (ODN) and Reagents

All ODN were synthesized following standard phosphoramidite chemistryprotocols and controlled for identity and purity by Coley PharmaceuticalGmbH and had undetectable endotoxin levels (<0.1 EU/ml) measured by theLimulus assay (BioWhittaker, Verviers, Belgium). ODN were suspended insterile, endotoxin-free Tris-EDTA (Sigma, Deisenhofen, Germany), andstored and handled under aseptic conditions to prevent both microbialand endotoxin contamination. All dilutions were carried out usingendotoxin-free Tris-EDTA.

TLR Assays

HEK293 cells were transfected by electroporation with vectors expressingthe respective human TLR and a 6×NF-κB-luciferase reporter plasmid.Stable transfectants (3×10⁴ cells/well) were incubated indicated amountsof ODN for 16 h at 37° C. in a humidified incubator. Each data point wasdone in triplicate. Cells were lysed and assayed for luciferase geneactivity (using the BriteLite kit from Perkin-Elmer, Zaventem, Belgium).Stimulation indices were calculated in reference to reporter geneactivity of medium without addition of ODN.

Cell Purification

Peripheral blood buffy coat preparations from healthy human donors wereobtained from the Blood Bank of the University of Düsseldorf (Germany)and PBMC were purified by centrifugation over Ficoll-Hypaque (Sigma).Cells were cultured in a humidified incubator at 37° C. in RPMI 1640medium supplemented with 5% (v/v) heat inactivated human AB serum(BioWhittaker) or 10% (v/v) heat inactivated FCS, 2 mM L-glutamine, 100U/ml penicillin and 100 μg/ml streptomycin (all from Sigma).

Cytokine Detection and Flow Cytometric Analysis

PBMC were resuspended at a concentration of 5×10 cells/ml and added to96 well round-bottomed plates (250 μl/well). PBMC were incubated withODN and culture supernatants (SN) were collected after the indicatedtime points. If not used immediately, SN were stored at −20° C. untilrequired.

Amounts of cytokines in the SN were assessed using an in-house ELISA forIFN-α developed using commercially available antibody (PBL, NewBrunswick, N.J., USA) or on the Luminex multiplex system (LumninexCorporation, 12212 Technology Boulevard, Austin, Tex. 78727-6115).

Animals

Female BALB/c mice (6-8 weeks of age) were purchased from Charles RiverCanada (Quebec, Canada) and housed in micro-isolators in the Animal CareFacility at Coley Pharmaceutical Group Canada. All studies wereconducted in accordance with the Animal Care Committee of Coley Canadaunder the guidance of the Canadian Council on Animal Care. All animalswere naïve to CpG ODNs.

SA1N tumor model: Female A/J mice (10 per group) were injected SC with5×10⁵ SaI/N tumor cells on day 0. Mice were treated with 100 μg ODN orPBS alone given SC once weekly starting on day 8 post tumor induction.Animals were monitored for survival and tumor volume. Tumor size (thelength and the width) was measured using a digital vernier caliper.Tumor volume was calculated by using the formula: Tumorvolume=(0.4)(ab2), where a=large diameter and b=smaller diameter.

In Vitro Assays

Naïve BALB/c mouse splenocytes (from pools of 3-5 animals) were used forin vitro assays. Animals were anaesthetized with isoflurane andeuthanized by cervical dislocation. Spleens were removed under asepticconditions and placed in PBS+0.2% bovine serum albumin (Sigma ChemicalCompany) Spleens were then homogenized and splenocytes were re-suspendedin RPMI 1640 (Life Technologies, Grand Island, N.Y.) tissue culturemedium supplemented with 2% normal mouse serum (Cedarlane Laboratories,Ontario, Canada), penicillin-streptomycin solution (final concentrationof 1000 U/ml and 1 mg/ml respectively, Sigma Chemical Company), and5×10−5 M b-mercaptoethanol (Sigma Chemical Company).

B Cell Proliferation Assays

Caboxy-florescein diacetate, succimidyl ester (CFSE) (Invitrogen,Eugene, Oreg., USA) stained BALB/c mouse splenocytes (4×10⁵/well) wereincubated with different concentrations of ODN in a humidified 5% CO₂incubator at 37° C. for 5 days. Cells were then stained with PEconjugated anti-CD19 antibody (BD Pharmingen, San Diego, Calif., USA)for CD19 and B-cell proliferation was determined by FACS followed byanalysis by ModFit Software V3.0 (Verity Software House Inc., Topsham,Me., USA).

Example 1 Investigation of Structure Activity Relationship at the CpGMotif

It is known that oligonucleotides containing unmethylated CpG motifs areable to stimulate immune responses through the Toll-like receptor 9(TLR9) pathway. In order to identify oligonucleotides with the greatestability to stimulate the TLR9 pathway, comprehensive structure activityrelationship (SAR) study at the CpG motif was performed. The resultsshowed that substitution of guanine by hypoxanthine and 6-thioguanineleads to a similar activity in hTLR9 assay, while purine, 2-aminopurine,2,6-diaminopurine, 8-oxo-7,8-dihydroguanine and 7-deazaguaninesubstitution resulted in a 40-80% reduction in hTLR9 stimulation.Further, modification at C5 and N4 resulted in no stimulation of thehTLR9 pathway. These observations resulted in a SAR model in whichguanine is recognized via the Hoogsteen site while cytosine binds at theC,H-Edge to the TLR9 receptor (see FIG. 1a ). Thus, no modification atthe Hoogsteen recognition site of guanine as well as the C,H-edge of thecytosine was possible without significant loss in hTLR9 activity. Noneof the investigated base modifications at the dinucleotide motif wasmore active than the unmodified CpG motif.

Example 2 The Effect of Hydrophobic Thymine Base Shape Analogs Near theCpG Motif

To investigate the impact of the dT residues in neighborhood to the CpGmotif several hydrophobic thymine base shape analogs, such as2,4-difluorotoluene (FF) (SEQ ID NO:3-9), 5-bromo-2′-deoxyuridine (BU)and 5-iodo-2′-deoxyuridine (JU), were incorporated outside of the CpGmotif (see Table 1 and FIGS. 2-3). Surprisingly, incorporation of alltested hydrophobic thymine analogs led to an unusually strong increasein hTLR9 activity, while substitution by uracil residues (thymine withlacking methyl group, FIG. 4) led to a sang decrease in hTLR9stimulation. The increase in TLR9 stimulation was pronounced when themodification was 5′ to the CpG motif Double substitution with5-iodouracil (JU) 5′ and 3′ of the CpG motif resulted in most potentstimulation of those tested. In contrast, substitution of guanine andcytosine by 2,4-difluorotoluene at the CpG motif led in both cases to astrong decrease of the TLR9 stimulation index.

Incorporation of hydrophobic T analogs also resulted in a strongenhancement of IFN-alpha induction in human PBMCs. Unexpectedly,modification of an ODN (SEQ ID NO:1) that is virtually inactive ininducing IFN-alpha with 5-bromouridine and 5-iodouridine in particularresulted in increased TLR9 stimulation and IFN-alpha induction. There isusually an inverse correlation between TLR9 and IFN-alpha induction forCpG ODN which do not contain these modifications.

TABLE 1 Examples of modified oligonucleotides with hydrophobicthymine base shape analogs near the CpG motif Seq ID Description/ No#Oligonucleotide sequence class derived from  1T*G*T*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T 1xPO of SEQ ID NO: 2  2T*G*T*C*G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T  3T*G*FF*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T 5′FF derivative of SEQ ID NO: 1 4 T*G*T*C-G*FF*T*T*T*T*T*T*T*T*T*T*T*T*T*T3′FF derivative of SEQ ID NO: 1  5T*G*FF*C-G*FF*T*T*T*T*T*T*T*T*T*T*T*T*T*T 3′ and 5′FF derivative of SEQ ID NO: 1  6 T*G*T*FF-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T C->FF  7T*G*T*C-FF*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T G->FF  8T*FF*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T GT->FF  9T*G*T*C-G*T*FF*T*T*T*T*T*T*T*T*T*T*T*T*T 3′FF derivative of SEQ ID NO: 110 T*G*BU*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T5′BU derivative of SEQ ID NO: 1 11T*G*T*C-G*BU*T*T*T*T*T*T*T*T*T*T*T*T*T*T 3′BU derivative of SEQ ID NO: 112 T*G*BU*C-G*BU*T*T*T*T*T*T*T*T*T*T*T*T*T*T 3′ and 5′BU derivative of SEQ ID NO: 1 13 T*G*JU*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T5′JU of SEQ ID NO: 1 14 T*G*T*C-G*JU*T*T*T*T*T*T*T*T*T*T*T*T*T*T3′JU of SEQ ID NO: 1 15 T*G*JU*C-G*JU*T*T*T*T*T*T*T*T*T*T*T*T*T*T 3′and 5′JU derivative of  SEQ ID NO: 1 16T*G*U*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T 5′U derivative of SEQ ID NO: 117 T*G*T*C-G*U*T*T*T*T*T*T*T*T*T*T*T*T*T*T3′U derivative of SEQ ID NO: 1 18T*G*U*C-G*U*T*T*T*T*T*T*T*T*T*T*T*T*T*T 3′ and 5′U derivative of SEQ ID NO: 1 *phosphorothioate internucleotide linkage -phosphodiesterinternucleotide linkage

Example 3 Activation of TLR9 with Lipophilic Base Shape Substitution

Since different types of lipophilic substitution of the base 5′ to theCpG motif caused significant increases in stimulation of hTLR9, otherbase analogs, such as 5-chloro-uracil, 5-trifluoromethyl-uracil, phenyl,aryl and substituted aryl residues were investigated for their abilityto stimulate hTLR9 (Table 3). To investigate activation of human TLR9 byB-class oligonucleotides modified with various lipophilic base analogs,B-class ODN SEQ ID NO:1 was modified with 5-Chloro-2′-deoxyuridine (CU),5-Bromo-2′-deoxyuridine (BU), 5-Iodo-2′-deoxyuridine (JU) and5-Ethyl-2′-deoxyuridine (EU). hTLR9-NFkB-293 cells were incubated withthe indicated ODN (FIG. 5a ) for 16 hours. Cells were then lysed andluciferase activity was determined. CU-modified (SEQ ID NO:41),BU-modified (SEQ ID NO:10) JU-modified (SEQ ID NO:13) and EU-modified(SEQ ID NO:42) oligonucleotides all showed greater stimulation of TLR9activity over control (SEQ ID NO:1). SEQ ID NO:16 with uridinemodification showed dramatically decreased activity. In a secondexperiment IFN-alpha production was measured (FIG. 5b ). Human PBMC wereincubated with the modified ODN as indicated for 24 h, after which thesupernatants were tested by ELISA. JU-modified, BU-modified, andEU-modified ODN resulted in the greatest increase in IFN-alpha overcontrol. These data demonstrate that 5′-substitution of dU on a B-classODN increases TLR9 activity and IFN-alpha production.

To further investigate the effect of EU modification on TLR9 activation,the experiment was repeated with modified oligonucleotides having EUmodifications 5′ of the CpG (SEQ ID NO:42). 3′ of the CpG (SEQ IDNO:29), and 5′ and 3′ of the CG (SEQ ID NO:30). SEQ ID NOs 42 and 30showed a significant increase in TLR9 activation over unmodified SEQ IDNO:1 and unmodified B class ODN SEQ ID NO:37 (FIG. 6).

TABLE 2Examples of modified oligonueleotides with lipophilie base analogsubstitutions Seq ID description/ No# Oligonucleotide sequenceclass derived from  1 T*G*T*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T Unmodified41 T*G*CU*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*TCI derivative of SEQ ID NO: 1 10T*G*BU*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T 5′BU derivative of SEQ ID NO: 113 T*G*JU*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T5′JU derivative of SEQ ID NO: 1 16T*G*U*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T U derivative of SEQ ID NO: 1 41T*G*CU*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T CU derivative of SEQ ID NO: 142 T*G*EU*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*TEU derivative of SEQ ID NO: 1 29T*G*T*C-G*EU*T*T*T*T*T*T*T*T*T*T*T*T*T*T 3′EU derivative of SEQ ID NO: 1 30T*G*EU*C-G*EU*T*T*T*T*T*T*T*T*T*T*T*T*T*T 5′3′EU derivative of SEQ ID NO: 1 *phosphorothioate internucleotide linkage-phosphodiester internucleotide linkage

Example 4 Lipophilic Substitution on Oligonucleotides of A, B, C, P, andT Classes

To investigate the effects of lipophilic base analog substitution on thedifferent classes of ODN, modifications were made on A class, B class, Cclass, P class, and T class oligonucleotides. Some examples of theseoligonucleotides are given in Table 3.

TABLE 3 JU-modified oligonucleotides of A B, C, P, and T class Seq IDNo. Modified Oligonucleotides Oligo Class 16T*G*U*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T B 17T*G*T*C-G*U*T*T*T*T*T*T*T*T*T*T*T*T*T*T B 18T*G*U*C-G*U*T*T*T*T*T*T*T*T*T*T*T*T*T*T B 19JU*C*G*T*C*G*T*T*T*T*T*C*G*G*T*C*G*T*T*T*T B 20T*C*G*JU*C*G*T*T*T*T*T*C*G*G*T*C*G*T*T*T*T B 21T*C*G*T*C*G*T*T*T*T*T*C*G*G*JU*C*G*T*T*T*T B 22JU*C*G*JU*C*G*T*T*T*T*T*C*G*G*T*C*G*T*T*T*T B 23T*C*G*JU*C*G*JU*T*T*T*T*C*G*G*T*C*G*T*T*T*T B 24T*C*G*T*C*G*T*T*T*T*T*C*G*G*JU*C*G*JU*T*T*T B 25T*C*T*T*T*T*T*T*G*T*C-G*T*T*T*T*T*T*T*T*T*T T 26T*G*C*T*G*C*T*T*T*T*G*T*G*C*T*T*T*T*G*T*G*C*T*T Non CpG ODN 27JU*C-G*T*C*G*T*T*T*T*A*C*G*G*C*G*C*C*G*T*G*C*C*G C 28T*C*G*JU*C-G*T*T*T*T*A*C*G*G*C*G*C*C*G*T*G*C*C*G C 31JU*C-G*T*C*G*A*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G P 32T*C*G*JU*C-G*A*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G P 33JU*C-G*JU*C*G*A*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G P 34JU*C-G-A-C-G-T-C-G-T-G-G*G*G*G A 35 T*C-G-A-C-G-JU-C-G-T-G-G*G*G*G A 36T*C-G-A-C-G-JU-C-G-JU-G-G*G*G*G A 37T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*C*G*T*T*T*T B 43T*C-G-A-C-G-T-C-G-T-G-G*G*G*G A 44JU*C-G*JU*C*G*T*T*T*T*A*C*G*G*C*G*C*C*G*T*G*C*C*G P 45T*C*G*JU*C-G*JU*T*T*T*A*C*G*G*C*G*C*C*G*T*G*C*C*G P 46T*C*G*T*C-G*T*T*T*T*A*C*G*G*C*G*C*C*G*T*G*C*C*G P 47T*C*T*T*T*T*T*T*G*JU*C-G*T*T*T*T*T*T*T*T*T*T T 48T*C*T*T*T*T*T*T*JU*C-G*JU*T*T*T*T*T*T*T*T*T T 49JU*C*T*T*T*T*T*T*G*T*C-G*T*T*T*T*T*T*T*T*T*T T 50JU*C-T*T*T*T*T*TG*T*C-G*T*T*T*T*T*T*T*T*T*T T 51T*C*T*T*T*T*T*T*G*U*C-G*T*T*T*T*T*T*T*T*T*T T 52T*C-G*T*C*G*A*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G P -pliosphodiesterinternuclentide linkage *phosphorothioate internuclectide linkage

To investigate activation of human TLR9 by modified B classoligonucleotides, 5-iodo-2′-deoxyuridine-modified B-class derivatives ofSEQ ID NO:3 were evaluated in a luciferase assay for their ability toactivate TLR9 (see materials and methods). All modified B-classoligonucleotides showed a significant increase in TLR9 activation overunmodified SEQ ID NO:37 (FIG. 7).

To investigate activation of human TLR9 by modified A-classoligonucleotides, 5-iodo-2′-deoxyuridine-modified A-class derivatives ofSEQ ID NO:43 were tested for their ability to activate TLR9 in aluciferase assay (FIG. 8a ) and a PBMC assay (FIG. 8b ) as in FIG. 5.The increase in TLR9 stimulation was pronounced when the modificationwas 5′ to the CpG motif; although double substitution with 5-iodouracil(JU) 5′ and 3′ of the CpG motif resulted in most potent stimulation.

To investigate the activation of human TLR9 by modified C classoligonucleotides, 5-iodo-2′-deoxyuridine-modified C class derivatives ofSEQ ID NO:46, SEQ ID NO:44 and 45, were tested for their ability toactivate TLR9. A class sequences SEQ ID NO:43 (unmodified) and SEQ IDNO:35 and 36 were tested simultaneously. As shown in FIG. 9, modifiedODN SEQ ID NO:35, 36, 44, and 45 all showed increased stimulation ofTLR9 above unmodified A and C class in a luciferase assay. Toinvestigate the activation of human TLR9 by modified P classoligonucleotides, 5-iodo-2′-deoxyuridine-modified P class derivatives ofSEQ ID NO:46 were tested for their ability to activate TLR9 in aluciferase assay. As shown in FIG. 10, modified ODN SEQ ID NO:31-33showed a increased stimulation of TLR9 over unmodified ODN.

To investigate the activation of human TLR9 by modified T classoligonucleotides, 5-iodo-2′-deoxyuridine-modified T class derivatives ofunmodified T class ODN SEQ ID NO:52 were tested for their ability toactivate TLR9. As shown in FIG. 11, modified ODN SEQ ID NOs 47-50 showedan increased stimulation of TLR9 over unmodified T class ODN in aluciferase assay. The uridine derivative SEQ ID NO:51 showed reducedstimulation of TLR9.

As the above examples demonstrate, substitution of lipophilic T-analogs5′ to the CpG motif results in a strong increase in TLR9 activation inall classes tested, and resulted in an increased ability to induceIFN-alpha production.

Example 5 Stimulation of TLR9 by Short Modified Oligonucleotides

As the modified CpG ODN of 20 nucleotides in length showed an unusualaffinity for TLR9 activation, very short CpG ODN were investigated fortheir ability to activate TLR9. Very short oligonucleotides would be agreat advantage over longer oligonucleotides for use in treatmentbecause of the increased ease in uptake by cells, as well as thepotential a simpler formulation, without the use of DOTAP. Three shortCpG ODN (shortmers) were investigated (Table 3): a 6-mer CpG motifhexamer (SEQ ID NO:38), a 5′JU modification of the hexamer (SEQ IDNO:39), and a 5′3′ JU modification of the hexamer (SEQ ID NO:40) (Table4). The activity of the shortmers was compared to the unmodified B classoligonucleotide SEQ ID NO:37 in a luciferase assay. As shown in FIG. 12,most particularly with SEQ ID NO:40, the use of modified shortmers showsgreat potential as an improved immunotherapy medicament.

TABLE 4 Modified short oligonucleotides SEQ ID No. Shortmer sequenceModification 38 G*T*C-G*T*T Unmodified 39 G*JU*C-G*T*T 5′ JU 40G*JU*C-G*JU*T 5′ and 3′ JU 37 T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*C*G*T*T*T*TUnmodified B class *phosphorothioate internucleolide linkage-phosphodiester internucleofide linkage

Example 6 Activation of TLR9 Pathway In Vivo by Modified Oligonucleotide

In order to determine the efficacy of the modified ODN of the inventionin vivo, ODN with lipophilic T analogs were tested in isolated mousesplenocytes. BALB/c mouse splenocytes were isolated and incubated withmodified B class (SEQ ID NO:13), unmodified B class (SEQ ID NO:37), anda non-CpG ODN (SEQ ID NO:26) (Table 5). Culture supernatants werecollected at 6 hour (TNF-alpha) or 24 hours (IL-6, IL-10, IL-12) andcytokine concentration was measured by ELISA. As shown in FIG. 13,incubation with modified SEQ ID NO:13 resulted in dramatically increasedlevels of all cytokines tested.

ODN were then tested their ability to induce B cell proliferation insplenocytes. CFSE-stained BALB/c mouse splenocytes (4×10⁵/well) wereincubated with 0.001, 0.01, 0.1, 0.3, 1, 3 or 10 μg/ml of the indicatedODN (FIG. 14). At 72 hours post-incubation, cells were stained for cellsurface marker CD19 and B-cell proliferation was determined by FACSfollowed by analysis by ModFit Software. As shown in FIG. 14, incubationwith modified SEQ ID NO:13 resulted in a marked increase in B-cellproliferation. The increase was most pronounced even at lower ODNconcentration.

To measure the effect of modified ODN in vivo, BALB/c mice (5 per group)were injected subcutaneously (SC) with 10, 50 or 100 μg of SEQ ID NO:13or 100 μg of SEQ ID NO:37 in a total volume of 100 μl SC. Control groupreceived 100 μl of PBS alone. Animals were bled by cardiac puncture at 1hour post injection (TNF-alpha) or 3 hours post injection (IP-10).Plasma samples were assayed ELISA for TNF-alpha (FIG. 15a ) and IP-10(FIG. 15b ). Injection of BALB/c mice with modified SEQ ID NO:13resulted in higher TNF-alpha and IP-10 production than the non-modifiedSEQ ID NO:37, demonstrating that the lipophilic base shape substitutedODN of the invention result in greater immune stimulation in vivo thanunmodified immune stimulatory ODN.

TABLE 5 Oligonucleotides tested in vivo Seq ID No. Sequence Modification13 T*G*JU*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T 5′ JU derivative of SEQ ID NO: 1 37 T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*C*G*T*T*T*TUnmodfified B class 26 T*G*C*T*G*CC*T*T*T*T*G*T*G*C*T*T*T*T*G*T*G*C*T*TNon CpG control *phosphorothioate intemucleotide linkage -phosphodiesterintemucleotide linkage

Example 7 Oligonucleotides with Additional Modifications

ODN with lipophilic base analogs were tested for their ability to induceTLR9-mediated NF-κB activity in a luciferase assay (see materials andmethods). FIGS. 16-23 show the activity of ODN with additionalmodifications (see table 6).

In order to test the activity of other base analogs, the activity of6-nitrobenzimidazol (6NB)-modified ODN SEQ ID NO:178 and unmodifiedparent sequence SEQ ID NO:1 was compared. As shown in FIG. 12, SEQ IDNO:178 was able to activate TLR9-mediated NF-κB to a degree comparablewith the unmodified parent sequence. Next the activity of5-(2-bromovinyl)-uridine modified ODN (SEQ ID NO:153-154) was comparedto that of unmodified parent sequence SEQ ID NO:1. As shown in FIG. 17,both modified ODN were more active in the assay than the parentsequence. Neat the activity of two B-class ODN with 5-proynyl-dU (SEQ IDNO:116 and 117) in place of thymidine of the parent sequence (SEQ IDNO:1). As shown in FIG. 21, both modified ODN had activity comparable tothat of the parent sequence. The activity of SEQ ID NO:116, in which themodification is 5′ to the CG dinucleotide, was slightly improved overthe parent sequence.

In order to test the effect of a second type of modification onJU-modified ODN, 2′O-methylguanosines were incorporated into JU-modifiedODN. The activity of 2′-O-methylguanosine/JU ODN SEQ ID NO:111-113 wascompared to that of parent SEQ ID NO:1 and JU only modified SEQ IDNO:13. As shown in FIG. 18, all JU-modified ODN were more active thanthe parent ODN. ODN with the 2′O-methylguanosine modification 3′ of theCG dinucleotide (SEQ ID NO:112-113) were slightly more active than theODN with the 2′O-methylguanosine modification 5′ of the CG dinucleotide(SEQ ID NO:111) or the ODN modified with JU alone (SEQ ID NO:13).

Next the activity of the JU-modified branched ODN (SEQ ID NO:96, 97,101, and 102) was compared to that of SEQ ID NO:1. As shown in FIG. 19,the branched ODN with two accessible 5′ ends were all as active or moreactive than the unmodified SEQ ID NO:1 in the assay. SEQ ID NO:101 and102, with the triethyleneglycol phosphate spacer, were more active thanSEQ ID NO:96 AND 97 with the 3′-O-Methyl-G spacer.

Next the activity of a short unmodified B-class ODN (SEQ ID NO:38) andan ODN of the same sequence with a lipophilic substituted nucleotideanalog and a lipophilic 3′ tag (SEQ ID NO:126) was compared. Both wereformulated with and without DOTAP. As shown in FIG. 20, the addition ofthe JU-modification and the lipophilic tag greatly enhanced the activityof the ODN, as did the addition of DOTAP.

Next the activity of B-class ODN with a second nucleotide analog inaddition to a lipophilic substituted nucleotide analog (SEQ ID NO:138,7-deaza-dG; SEQ ID NO:139, inosine; SEQ ID NO:140, 5-methyl-dC) wascompared to that of the parent sequence (SEQ ID NO:1) and the samesequence with a lipophilic substituted nucleotide analog only (SEQ IDNO:13). As shown in FIG. 22, all modified ODN were more active in theassay than the parent ODN

Next the activity of T-class ODN with a lipophilic substitutednucleotide analog (SEQ ID NO:132-134) was compared to that of a C-classODN (SEQ ID NO:198) known to be immunostimulatory. As shown in FIG. 23,all modified ODN showed much greater activity in the assay than theunmidified C-class ODN.

TABLE 6Lipophilic substituted oligonuclotides with additional modificationsSeq ID No. Sequence Type and modfication   1T*G*T*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T unmodified B-class  13T*G*JU*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T B-class: 5′JU derivative ofSEQ ID NO: 1  38 G*T*C-G*T*T Unmodified B-class  96(T*G*JU*C-G*T*T*L*)2doub-3mg 3′3′-branched  97(JU*C*G*T*T*C*G*L*)2doub-3mg 3′3′-branched 101(T*G*JU*C-G*T*T*L*)2doub-teg 3′3′-branched 102(JU*C*G*T*T*C*G*L*)2doub-teg 3′3′-branched 111T*mG*JU*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T 2′-O-methyl-modified B-class112 T*G*JU*C-mG*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T2′-O-methyl-modified B-class 113T*mG*JU*C-mG*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T 2′-O-methyl-modified B-class116 T*G*PU*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*TB-class with 5-proynyl-dU (PU) 117T*G*T*C-G*PU*T*T*T*T*T*T*T*T*T*T*T*T*T*T B-class with 5-proynyl-dU (PU)126 G*JU*C-G*JU*T-hex B-class derivative of 38 withJU and hexadaclglyceryl 3′ tag 132JU*C*T*T*T*T*T*T*T*T*C*G*T*T*T*T*T*T*T*T*T*T T-class 133T*C*T*T*T*T*T*T*T*JU*C*G*T*T*T*T*T*T*T*T*T*T T-class 134JU*C*T*T*T*T*T*T*T*JU*C*G*T*T*T*T*T*T*T*T*T*T T-class 138T*G*JU*C-E*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T B-class with 7-deaza-dG (E) 139T*G*JU*C-I*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T B-class with inosine (I) 140T*G*JU*Z-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T B-class with 5-metyl-dC (Z) 153T*G*BVU*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*TB-class with 5-(2-bromo-vinyl)- uridine (BVU) 154T*G*T*C-G*BVU*T*T*T*T*T*T*T*T*T*T*T*T*T*TB-class with 5-(2-bromo-vinyl)- uridine (BVU) 178T*G*BNB*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T B-class with 6-nitro-benzimidazol (6NB) 198 C*G*G*C*G*C*C*T*C*G C-class *phosphorothloateinternueleotide linkage -phosphodiester internueleotide linkage

Example 8 Activity of Modified P-Class Oligonucleotides

P-class ODN with lipophilic base analogs were tested for the ability toactivate the NF-kB pathway through TLR9 as measured by luciferase assay.The activity of P-class ODN with a lipophilic substituted nucleotideanalog (SEQ ID NO:58-61) was compared to that of a B-class positivecontrol (SEQ ID NO:55) and an unmodified P-class ODN (SEQ ID NO:56). Asshown in FIG. 24, all modified P-class ODN showed increased TLR9stimulation compared to the controls. FIG. 24a shows JU-modified P-classODN and 24 b shows EU-modified P-class ODN.

Next the activity of modified P-class ODN (SEQ ID NO:64 (EU-modified),66-67 (JU-modified) was compared to that of a B-class positive control(SEQ ID NO:55), a C-class ODN (SEQ ID NO:68) and an unmodified P-classODN (SEQ ID NO:57). As shown in FIG. 25, all modified ODN showed ahigher degree of TLR9 stimulation than the unmodified P class ODN. SEQID NO:66, with the phosphodiester bond in the CG dinucleotide, showedreduced activity compared to the fully phosphorothioate SEQ ID NO:67.

Next the modified P-class ODN were tested for their ability to induceexpression of IFN-alpha. The activity of P-class ODN with a lipophilicsubstituted nucleotide analog (SEQ ID NO:58-61) was compared to that ofa B-class positive control (SEQ ID NO:55) and an unmodified P-class ODN(SEQ ID NO:56) as measured by an ELISA assay. As shown in FIG. 26, allmodified P-class ODN showed an increase in IFN-alpha induction. FIG. 26ashows JU-modified P-class ODN and 26 b shows EU-modified P-class ODN.

Next the modified P-class ODN (EU-modified), 66-67 (JU-modified) wascompared to that of a B-class positive control (SEQ ID NO:55), a C-classODN (SEQ ID NO:68) and an unmodified P-class ODN (SEQ ID NO:57) for theability to induce IFN-alpha as measured by an ELISA assay. As shown inFIG. 27, the modified P-class ODN showed enhanced ability to induceIFN-alpha. As in FIG. 24, SEQ ID NO:66 showed reduced activity comparedto SEQ ID NO:67.

Next the modified P-class ODN were tested for the ability to induce IL-6in human PBMC. PBMC from three donors were incubated with ODN atconcentrations as indicated for 24 h, followed by luminex 25-plexanalysis of the supernatants for IL-6. The activity of modified P-classODN (SEQ ID NO:58, 60-62, FIG. 28a ) (SEQ ID NO:64 and 67, FIG. 28b )was compared to that of an unmodified B-class ODN (SEQ ID NO:55), andunmodified C-class ODN (SEQ ID NO:54), a negative control ODN (SEQ IDNO:53), and an unmodified P-class ODN (SEQ ID NO:56). The JU-modifiedODN (SEQ ID NO:58, 60-61 and 67) showed a slightly higher activation ofIL-6 than did the EU-modified ODN (SEQ ID NO:62 and 64). All modifiedODN showed increased activity compared to unmodified ODN.

Next the activity of modified P-class class ODN (SEQ ID NO 58, 60-62,FIG. 29a ) (SEQ ID NO:64 and 67, FIG. 29b ) was compared to that of anunmodified B-class ODN (SEQ ID NO:55), an unmodified C-class ODN (SEQ IDNO:54), a negative control ODN (SEQ ID NO:53), an unmodified P-class ODN(SEQ ID NO:56) LPS, R-848, SEB, and a poly[I]:[C] ODN. CFSE-labeled PBMCfrom three donors were incubated with the ODN for 5 days and thenstained with a CD19 antibody. The percentage of B cells with reducedCFSE staining was determined. Treatment with the B-class ODN resulted inthe highest percentage of B cells after division. Treatment with theJU-modified ODN resulted in a higher percentage of B cells than theEU-modified ODN.

In order to determine the effect of the modified P-class ODN in vivo,BALB/c mice (5 per group) were injected SC with differing doses of ODN.Animals were bled at 3 hr post injection and plasma tested for IFN-alphaby ELISA. The activity of modified P-class ODN(SEQ ID NO:58, 60-62, 64,and 67) was compared to that of a B-class negative control (SEQ IDNO:55) and a negative control (SEQ ID NO:26). As shown in FIG. 30,treatment with the JU-modified ODN SEQ NO:58, 60, and 61 resulted inslightly higher IFN-alpha induction than the EU-modified ODN SEQ IDNO:64. The B-class ODN SEQ ID NO:55 did not induce much murineIFN-alpha, as expected.

Next the modified P-class ODN were evaluated for their ability to reducetumor volume mouse SA1N tumor model. Female A/J mice (10 per group) wereinjected SC with 5×10⁵ SaI/N tumor cells an day 0. Mice were treatedwith 35 g (FIG. 31a ) or 100 μg (FIG. 31b ) P-class ODN with alipophilic substituted nucleotide analog (SEQ ID NO:60, 64, and 67), anunmodified C-class ODN, an unmodified B-class ODN (SEQ ID NO:55), or PBSalone. ODN were given SC once weekly starting on day 8 post tumorinduction. Animals were monitored for survival and tumor volume. Asshown in FIG. 31a , at the lower dosage treatment with the modifiedP-class ODN showed the greatest reduction in tumor volume, suggestingthat these ODN would be effective in treating cancer. At the higherdosage in 31 b, all modified P-class ODN and the C-class ODN wereeffective in reducing tumor volume.

TABLE 7 Modified P-class oligonucleotides Seq ID Type and No. Sequencemodification 53 T*C*C*A*G*G*A*C*T*T*C*T*C*T*C*A*G*G*T*T neg control 54T*C*G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G C-class 55T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T B-class 56T*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G P-class T->A, 5′ CpG PO 57T*C-G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C*G P-class 3′ palin-drome, 5′ CpG PO 58 JU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*GP-class 59 JU*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G P-class 60JU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G*T P-class 61JU*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G*T P-class 62EU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G P-class 63EU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G P-class 64EU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G P-class 65EU*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G P-class 66JU*C-G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C*G*T P-class 67JU*C*G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C*G*T P-class 68T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G C-class *phosphorothloateinternueleotide linkage -phosphodiester internueleotide linkage

A summary of Exemplary modified ODN is presented in Table 8:

TABLE 8 Seq ID No# Oligonucleotide sequence   3T*G*FF*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T   4T*G*T*C-G*FF*T*T*T*T*T*T*T*T*T*T*T*T*T*T   5T*G*FF*C-G*FF*T*T*T*T*T*T*T*T*T*T*T*T*T*T   6T*G*T*FF-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T   7T*G*T*C-FF*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T   8T*FF*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T   9T*G*T*C-G*T*FF*T*T*T*T*T*T*T*T*T*T*T*T*T  10T*G*BU*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T  11T*G*T*C-G*BU*T*T*T*T*T*T*T*T*T*T*T*T*T*T  12T*G*BU*C-G*BU*T*T*T*T*T*T*T*T*T*T*T*T*T*T  13T*G*JU*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T  14T*G*T*C-G*JU*T*T*T*T*T*T*T*T*T*T*T*T*T*T  15T*G*JU*C-G*JU*T*T*T*T*T*T*T*T*T*T*T*T*T*T  16T*G*U*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T  17T*G*T*C-G*U*T*T*T*T*T*T*T*T*T*T*T*T*T*T  18T*G*U*C-G*U*T*T*T*T*T*T*T*T*T*T*T*T*T*T  19JU*C*G*T*C*G*T**T*T*T*T*C*G*G*T*C*G*T*T*T*T  20T*C*G*JU*C*G*T*T*T*T*T*C*G*G*T*C*G*T*T*T*T  21T*C*G*T*C*G*T*T*T*T*T*C*G*G*JU*C*G*T*T*T*T  22JU*C*G*JU*C*G*T*T*T*T*T*C*G*G*T*C*G*T*T*T*T  23 T*C*G*JU*C*G*JU*T*T*T*T*C*G*G*T*C*G*T*T*T*T  24T*C*G*T*C*G*T*T*T*T*T*C*G*G*JU*C*G*JU*T*T*T  27JU*C-G*T*C*G*T*T*T*T*A*C*G*G*C*G*C*C*G*T*G*C*C*G  28T*C*G*JU*C-G*T*T*T*T*A*C*G*G*C*G*C*C*G*T*G*C*C*G  29T*G*T*C-G*EU*T*T*T*T*T*T*T*T*T*T*T*T*T*T  30T*G*EU*C-G*EU*T*T*T*T*T*T*T*T*T*T*T*T*T*T  31JU*C-G*T*C*G*A*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G  32T*C*G*JU*C-G*A*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G  33JU*C-G*JU*C*G*A*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G  34JU*C-G-A-C-G-T-C-G-T-G-G*G*G*G  35 T*C-G-A-C-G-JU-C-G-T-G-G*G*G*G  36T*C-G-A-C-G-JU-C-G-JU-G-G*G*G*G  39 G*JU*C-G*T*T  40 G*JU*C-G*JU*T  41T*G*CU*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T  42T*G*EU*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T  44JU*C-G*JU*C*G*T*T*T*T*A*C*G*G*C*G*C*C*G*T*G*C*C*G  45T*C-G*JU*C*G*JU*T*T*T*A*C*G*G*C*G*C*C*G*T*G*C*C*G  47T*C*T*T*T*T*T*T*G*JU*C-G*T*T*T*T*T*T*T*T*T*T  48T*C*T*T*T*T*T*T*G*JU*C-G*JU*T*T*T*T*T*T*T*T*T  49JU*C*T*T*T*T*T*T*G*T*C-G*JU*T*T*T*T*T*T*T*T*T*T  50JU*C-T*T*T*T*T*T*G*T*C-G*T*T*T*T*T*T*T*T*T*T  51T*C*T*T*T*T*T*T*G*U*C-G*T*T*T*T*T*T*T*T*T*T  58JU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G  59JU*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G  60JU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G*T  61JU*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G*T  62EU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G  63EU*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G  64JU*C-G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C*G*T  65JU*C*G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C*G*T  66EU*C-G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C*G  67JU*C-G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C*G  78T*G*T*C-G*FU*T*T*T*T*T*T*T*T*T*T*T*T*T*T  79T*G*FU*C-G*FU*T*T*T*T*T*T*T*T*T*T*T*T*T*T  80T*G*Y*C-G*U*T*T*T*T*T*T*T*T*T*T*T*T*T*T  81T*G*T*C-6NB*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T  82T*G*T*6NB-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T  83T*G*T*6NB-G-T*T*T*T*T*T*T*T*T*T*T*T*T*T*T  84JU*G*T*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T  85JU*G*JU*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T  86T*G*T*C-G*T*JU*T*T*T*T*T*T*T*T*T*T*T*T*T  87T*G*FT*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T  88T*G*T*C-G*FT*T*T*T*T*T*T*T*T*T*T*T*T*T*T  89T*G*FT*C-G*FT*T*T*T*T*T*T*T*T*T*T*T*T*T*T  90T*G*CU*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T  91T*G*T*C-G*CU*T*T*T*T*T*T*T*T*T*T*T*T*T*T  92T*G*CU*C-G*CU*T*T*T*T*T*T*T*T*T*T*T*T*T*T  93T*JU*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T  94 T*G*JU*C-G*T*T*T*T  95T*G*JU*C-G*T*T*T*T*G*T*C-G*T*T  96 (T*G*JU*C-G*T*T*L*)2doub-3mG  97(JU*C*G*T*T*C*G*L*)2doub-3mG  98 T*T*JU*C-G*T*C-G*T*T*T*C-G*T*C-G*T*T 99 BU*C-G-A-C-G-T-C-G-T-G-G-G*G*G 100T*G*JU*G-C*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T 101 (T*G*JU*C-G*T*T*L*) 102(JU*C*G*T*T*C*G*L*)2doub-teg 103JU*C-G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G 104T*C*G*JU*C-G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G 105T*C*G*T*C*G*T*T*T*JU*C-G*G*C*G*C*G*C*G*C*C*G 106JU*C*G*T*C*G*T*T*T*T*T*T*C*G*G*JU*C*G*T*T*T*T 107T*C*G*JU*C*G*T*T*T*T*T*C*G*G*JU*C*G*T*T*T*T 108T*G*JU*C-G*T*T*T*T*T*T*T*T*T*G*JU*C-G*T*T 109T*G*JU*C*G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T 110JU*C-G-A-C-G-T-C-G-T-G-G*E*G*G 111T*mG*JU*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T 112T*G*JU*C-mG*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T 113T*mG*JU*C-mG*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T 114JU*C-G*JU*C*G*T*T*T*T*T*C*G*G*T*C*G*T*T*T*T 115JU*C*G*JU*C-G*T*T*T*T*T*C*G*G*T*C*G*T*T*T*T 116T*G*PU*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T 117T*G*T*C-G*PU*T*T*T*T*T*T*T*T*T*T*T*T*T*T 118BU*C-G-A-C-G-T-C-G-T-G-G*G*G*G 119T*G*JU*C-G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G 120T*JU*C-G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G*T 121T*EU*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T 122T*G*EU*G-C*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T 123JU*C-G*T*C*G*T*T*T*T*T*C*G*G*T*C*G*T*T*T*T 124EU*C-G*T*C*G*T*T*T*T*T*C*G*G*T*C*G*T*T*T*T 125 G*JU*C-G*T*T*-hex 126G*JU*C-G*JU*T-hex 127 G*EU*C-G*EU*T-hex 128EU*C-G*T*C*G*T*T*T*T*A*C*G*G*C*G*C*C*G*T*G*C*C*G 129T*C*G*EU*C-G*T*T*T*T*A*C*G*G*C*G*C*C*G*T*G*C*C*G 130EU*C-G*T*C*G*A*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 131JU*C*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T 132JU*C*T*T*T*T*T*T*T*T*C*G*T*T*T*T*T*T*T*T*T*T 133T*C*T*T*T*T*T*T*T*JU*C*G*T*T*T*T*T*T*T*T*T*T 134JU*C*T*T*T*T*T*T*T*JU*C*G*T*T*T*T*T*T*T*T*T*T 135JU*C-G*T*C*G*T*T*T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T* 136T*C*G*T*C*G*T*T*T*C*G*T*C*G*T*T*T*T*G*JU*C-G*T*T 137JU*C-G*T*C*G*T*T*T*C*G*T*C*G*T*T*T*T*G*JU*C-G*T*T 138T*G*JU*C-E*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T 139T*G*JU*C-I*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T 140T*G*JU*C-Z*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T 141T*G*T*C-G*T*T*JU*T*T*T*T*T*T*T*T*T*T*T*T 142T*G*T*C-G*T*T*T*JU*T*T*T*T*T*T*T*T*T*T*T 143JU*C-G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G*T 144EU*C-G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G*T 145T*C-G*EU*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G*T 146T*C-G*T*C*G*T*T*T*JU*C*G*G*C*G*C*G*C*G*C*C*G*T 147T*C-G*T*C*G*T*T*T*EU*C*G*G*C*G*C*G*C*G*C*C*G*T 148EU*C-G*T*C*G*T*T*T*EU*C*G*G*C*G*C*G*C*G*C*C*G*T 149EU*C-G*EU*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G*T 150JU*C-G*EU*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G*T 151JU*C-G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T 152EU*C-G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T 153T*G*BVU*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T 154T*G*T*C-G*BVU*T*T*T*T*T*T*T*T*T*T*T*T*T*T 155 JU*C*G*G*C*G*G*C*C*G*C*C*G156 JU*C*G*T*C*G*T*T*T*T*A*C*G*G*C*G*C*C*G*T*G*C*C*3mG 157EU*C*G*T*C*G*T*T*T*T*A*C*G*G*C*G*C*C*G*T*G*C*C*3mG 158EU*C*G*EU*C*G*T*T*T*T*A*C*G*G*C*G*C*C*G*T*G*C*C*3mG 159EU*C-G*EU*C*G*T*T*T*T*A*C*G*G*C*G*C*C*G*T*G*C*C*3mG 160EU*C*G*T*C*G*T*T*T*T*A*C*G*G*C*G*C*C*G*T*G*C*C*G*iT 161JU*C*G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*3mG 162EU*C*G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*3mG 163EU*C-G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*3mG 164EU*C*G*EU*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*3mG 165EU*C-G*EU*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*3mG 166EU*C*G*T*C*G*T*T*T*EU*C*G*G*C*G*C*G*C*G*C*C*3mG 167JU*C*G*T*C*G*T*T*T*JU*C*G*G*C*G*C*G*C*G*C*C*3mG 168EU*C*G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G*iT 169JU*C*G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G*iT 170EU*C*G*T*C*G*A*C*G*T*T*C*G*G*C*G*C*C*G*T*G*C*C*3mG 171JU*C*G*T*C*G*A*C*G*T*T*C*G*G*C*G*C*C*G*T*G*C*C*3mG 172JU*C*G*T*C*G*A*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*3mG 173EU*C*G*T*C*G*A*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*3mG 174EU*C*G*T*C*G*A*C*G*T*T*C*G*G*C*G*C*C*G*T*G*C*C*G*iT 175EU*C*G*T*C*G*A*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G*iT 176T*G*bi*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T 177T*G*NP*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T 178T*G*6NB*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T 179EU*C*G*T*C*G*T*T*T*T*T*C*G*G*T*C*G*T*T*T*T 180JU*C*G*T*C*G*A*C*G*A*T*G*G*C*G*G*C*G*C*C*G*C*C 181EU*C*G*T*C*G*A*C*G*A*T*G*G*C*G*G*C*G*C*C*G*C*C 182T*T*C-G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G*T 183T*EU*C-G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G*T 184JU*C-G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G*T 185JU*JU*C-G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G*T 186T*JU*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G*T 187EU*C*G*T*C*G*T*T*T*T*A*C*G*G*C*G*C*C*G*T*G*C*C*G*T 188T*EU*C*G*T*T*T*T*A*C*G*G*C*G*C*C*G*T*G*C*C*G*T 189T*JU*C*G*T*T*T*T*A*C*G*G*C*G*C*C*G*T*G*C*C*G*T 190JU*C*G*T*C*G*T*T*T*T*rG*rU*rU*rG*rG*rU 191EU*C-G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C*G*T 192EU*C*G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C*G*T 193EU-C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 194EU-C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 195T*G*U*C-G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T 196T*G*T*C-G*U*T*T*T*T*T*T*T*T*T*T*T*T*T*T

EQUIVALENTS

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention.

We claim:
 1. An oligonucleotide comprising: the sequence R₁CGR₂, wherein R₁ and R₂ are selected from the group consisting of a lipophilic substituted nucleotide analog (L), a nucleotide, and a linkage; wherein at least one of R₁ and R₂ is a lipophilic substituted nucleotide analog (L), wherein the nucleobase ring of L is selected from the group consisting of 5-chloro-uracil, 5-bromo-uracil, 5-ethyl-uracil, and (E)-5-(2-bromovinyl)-uracil; wherein at least two nucleotides of the oligonucleotide have a stabilized linkage; and wherein the stabilized linkage is a phosphorothioate, a phosphorodithioate, a methylphosphonate, a methylphosphonothioate, a boranophosphonate, a phosphoramidate, or a dephospho linkage, either as an enantiomeric mixture or as an enantiomeric pure S- or R-configuration.
 2. The oligonucleotide of claim 1, wherein R₁ is L and R₂ is a nucleotide.
 3. An oligonucleotide comprising: the sequence R₁CGR₂, wherein R₁ and R₂ are selected from the group consisting of a lipophilic substituted nucleotide analog (L), and a linkage; wherein at least one of R₁ and R₂ is a lipophilic substituted nucleotide analog (L), wherein the nucleobase ring of L is selected from the group consisting of 5-chloro-uracil, 5-bromo-uracil, 5-ethyl-uracil, and (E)-5-(2-bromovinyl)-uracil; and wherein R₁ is a L and R₂ is a linkage, such that the oligonucleotide comprises a structure 5′LCG3′, wherein L is the 5′ terminal nucleotide.
 4. The oligonucleotide of claim 1, wherein the oligonucleotide is 7-100 nucleotides in length.
 5. The oligonucleotide of claim 1, wherein the oligonucleotide comprises one to four unmethylated CG dinucleotides.
 6. The oligonucleotide of claim 1, wherein the oligonucleotide comprises a non-nucleotidic modification, wherein the non-nucleotidic modification is selected from the group consisting of C₆-C₄₈-polyethyleneglycol, C₃-C₂₀-alkane-diol, C₃-C₁₈-alkylamino linker, C₃-C₁₈-alkylthiol linker, cholesterol, bile acid, saturated or unsaturated fatty acid, folate, a hexadecyl-glycerol or dihexadecyl-glycerol group, an octadecyl-glycerol or dioctadecylglycerol group, and a vitamin E group.
 7. The oligonucleotide of claim 1, wherein the stabilized linkage is a phosphorothioate.
 8. The oligonucleotide of claim 1, wherein the CG of R₁CGR₂ has a phosphodiester linkage.
 9. The oligonucleotide of claim 8, wherein all other nucleotides have a phosphorothioate linkage. 